Product and Methods for Diagnosis and Therapy for Cardiac and Skeletal Muscle Disorders

ABSTRACT

Disclosed are products and methods to promote myoblast activation and cardiac and skeletal muscle growth or regeneration, and to treat heart and skeletal muscle diseases, based on the identification of cellular processes affected by prelamin A processing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.10/803,541, filed Mar. 17, 2004, which claims the benefit of priorityunder 35 U.S.C. § 119(e) from U.S. Provisional Application No.60/456,642, filed Mar. 18, 2003, entitled “Product and Methods forDiagnosis and Therapy for Cardiac and Skeletal Muscle Disorders”. Theentire disclosure of both applications is incorporated herein byreference for all purposes.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “2848-53_ST25.txt”, having a size in bytes of 57 kb, andcreated on Mar. 17, 2004. The information contained in this electronicfile is hereby incorporated by reference in its entirety pursuant to 37CFR § 1.52(e)(5).

FIELD OF THE INVENTION

This invention generally relates to products and methods to promotemyoblast activation and cardiac and skeletal muscle growth orregeneration based on the identification of cellular processes affectedby prelamin A processing.

BACKGROUND OF THE INVENTION

Mutations in the human lamin A/C gene cause dilated cardiomyopathy (DCM)(Brodsky et al., (2000) Circulation 101:473-6; Fatkin et al., (1999) NEngl J Med 341:1715-24; Taylor et al., (2003) Journal of the AmericanCollege of Cardiology, 41:771-780), Emery-Dreifuss muscular dystrophy(Bonne et al., (1999) Nature Genet. 21:285-288), limb-girdle musculardystrophy (Muchir et al., (2000) Hum Mol Genet 9:1453-9), partiallipodystrophy (Shackleton et al., (2000) Nat Genet24:153-6), axonalneuropathy (De Sandre-Giovannoli et al., (2002) Am J Hum Genet70:726-36) and mandibuloacral dysplasia (Novelli et al., (2002) Am J HumGenet 71:426-31). Along with lamin B, lamins A and C are the majorconstituents of the nuclear lamina, a meshwork of protein filaments thatunderlies the nucleoplasmic face of the inner nuclear membrane. Thenuclear lamina provides structural support for the nucleus (Newport etal., (1990) J Cell Biol 111:2247-59; Spann et al., (1997) J Cell Biol136:1201-12), and plays a role in the regulation of gene transcriptionthrough direct and indirect interactions with transcription factors(Ozaki et al., (1994) Oncogene 9:2649-53; Markiewicz et al., (2002) MolBiol Cell 13:4401-13; Spann et al., (2002) J Cell Biol 156:603-8), andby organizing intranuclear RNA splicing factor compartments (Kumaran etal., (2002) J Cell Biol 159:783-93). Lamins bind directly to DNA, andare involved in chromatin organization via direct interactions withhistones and other chromatin binding proteins (Gotzmann & Foisner (1999)Crit Rev Eukaryot Gene Expr 9:257-65). Site-specific phosphorylation oflamin A results in the reversible disassembly of the lamina duringmitosis (Haas & Jost (1993) Eur J Cell Biol 62:237-47), and lamin A is atarget of endoproteolytic cleavage during apoptosis (Slee et al., (2001)J Biol Chem 276:7320-6).

Lamins A and C are differentially transcribed from the lamin A/C gene.Lamin A is expressed as a pre-protein (Gerace et al., (1984) J Cell SciSuppl 1: 137-60) that undergoes a sequential series ofpost-translational modifications (Sinensky et al., (1994) J Cell Sci107:61-7) shared by the S. cerevisiae a-type mating pheromone (Marcus etal., (1990) Biochem Biophys Res Commun 172:1310-6), culminating in theendoproteolytic removal of the modified 15 amino acid residue C-terminalpeptide. While proper processing of the prelamin A tail has been shownto affect the rate of mature lamin A incorporation into the nuclearlamina (Lutz et al., (1992) Proc Natl Acad Sci USA 89:3000-4; Izumi etal., (2000) Mol Biol Cell 11:4323-37), the physiological function ofprelamin A processing has not been determined.

Lamins A and C are expressed in nearly all cell types concomitant withdifferentiation (Rober et al., (1989) Development 105:365-78). Thereason why mutations in the lamin A/C gene result in tissue-specificabnormalities and the molecular mechanisms by which lamin A/C mutationsexert their effects on these tissues has yet to be elucidated.

The initial cloning of lamin A/C indicated that the protein wasprocessed. In the early 1990's, investigators began elucidating theprocessing pathway of prelamin A, and the localization andcharacteristics of enzymes involved in its processing (Lutz et al.,(1992), supra; Dalton et al., Cancer Res. 55:3295-3304 (1995); Sinenskyet al., J Cell Sci 107:2215-2218 (1994)). These researchers alsoinvestigated the biological function of the “pre” sequence by preventingits cleavage from the prelamin A protein, and inhibiting its processingin mononucleate cell lines. These studies demonstrated that the presenceof the “pre” sequence prevented incorporation into the lamina, and alsoshowed that mature lamin A lacking the pre-sequence could substitute forthe native prelamin A without any biological consequences. In one ofthese studies, the authors comment that “nucleoplasmic localization ofprelamin A or the peptide released during processing may have someregulatory significance” (Lutz et al., (1992), supra).

Investigators have reported the construction of a lamin A/C knock-outmouse that has cardiac and skeletal muscle phenotypes similar to thoseseen in patients with DCM and EDMD (Sullivan et al., (1999) J Cell Biol147:913-20). Over the last two years, investigators have generated micewhich lack the mouse homologues of the enzymes in the yeast Mat Aprocessing pathways (Pendas et al., Nat Genet 31:94-99 (2002); Bergo etal., (2002) Proc Natl Acad Sci USA 99:13049-54). These animals appear tobe phenocopies of the lamin A/C knock-out mice. These investigators havedemonstrated that prelamin A is not properly processed in these animals.

Consequently, the published literature demonstrates that lamin Aexpression, and proper prelamin A processing are essential for normalpost-natal cardiac and skeletal muscle biology in mice. The publisheddata also shows that prelamin A must be processed to mature lamin Aprior to incorporation into the nuclear lamina. However, there is nopublished data identifying the cellular function of prelamin Aprocessing, or the mechanism by which mutations in the lamin A/C gene,or the deletion of the lamin A/C gene and enzymes that process prelaminA, lead to cardiac and skeletal muscle abnormalities. Such informationwould be invaluable for in the understanding of cardiac and skeletalmuscle disease processes affected by lamin A/C disease mutations, aswell as the ability to design therapies to prevent these and othercardiac and skeletal muscle diseases.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to an isolated prelaminA pre peptide selected from: (a) a peptide consisting essentially of SEQID NO:2; (b) a biologically active fragment of SEQ ID NO:2; (c) apeptide consisting essentially of an amino acid sequence that is atleast about 70% identical to SEQ ID NO:2, wherein the peptide has thebiological activity of SEQ ID NO:2; and (d) a peptide consistingessentially of an amino acid sequence that differs from SEQ ID NO:2 byat least one substitution, deletion or insertion of an amino acidresidue at a position of SEQ ID NO:2 selected from the group consistingof: 1, 2, 5, 6, 9, 10, 11, 12, 13 and 14, wherein the peptide has thebiological activity of SEQ ID NO:2. In one aspect, the peptide consistsessentially of an amino acid sequence that is at least about 80%identical to SEQ ID NO:2, or in another aspect, is at least about 90%identical to SEQ ID NO:2. In one aspect, the peptide consistsessentially of an amino acid sequence that differs from SEQ ID NO:2 byat least one substitution, deletion or insertion of an amino acidresidue at a position of SEQ ID NO:2 selected from: 1, 2, 5, 6, 9, 10,11 and 12. In another aspect, the peptide consists essentially of anamino acid sequence that differs from SEQ ID NO:2 by at least onesubstitution, deletion or insertion of an amino acid residue at aposition of SEQ ID NO:2 selected from: 1, 2, 5, 6, 9, 10 and II. In oneaspect, the peptide consists essentially of SEQ ID NO:2. In anotheraspect, the peptide comprises a modification selected from the groupconsisting of farnesylation, carboxymethylation, geranyl-geranylation,and complexing with a lipid carrier.

Another embodiment of the present invention relates to a therapeuticcomposition comprising any of the above-described peptides and apharmaceutically acceptable carrier.

Yet another embodiment of the present invention relates to an isolatednucleic acid sequence encoding any of the above-described peptides. Inone aspect, the nucleic acid sequence is SEQ ID NO:1.

Another embodiment of the present invention relates to a recombinantnucleic acid molecule comprising any of the above-described nucleic acidsequences operatively linked to a recombinant vector. In one aspect, thenucleic acid sequence is operatively linked to a promoter selected from:a cardiac-specific promoter, a muscle-specific promoter, and a prelaminA promoter. In a preferred aspect, the nucleic acid sequence isoperatively linked to a myosin heavy chain promoter. In one aspect, therecombinant vector is a viral vector.

Yet another embodiment of the present invention relates to a recombinantnucleic acid molecule comprising a nucleic acid sequence operativelylinked to a recombinant expression vector for gene delivery, the nucleicacid sequence being selected from: (a) a nucleic acid sequence encodingSEQ ID NO:4; (b) a nucleic acid sequence encoding a biologically activefragment of SEQ ID NO:4; and (c) a nucleic acid sequence encoding anamino acid sequence that is at least about 70% identical to SEQ ID NO:4,wherein the amino acid sequence has prelamin A or lamin A biologicalactivity.

Another embodiment of the present invention relates to a therapeuticprotein comprising a protein selected from: (a) a protein comprising anamino acid sequence represented by SEQ ID NO:4; (b) a protein comprisingbiologically active fragment of SEQ ID NO:4; and (c) a proteincomprising an amino acid sequence that is at least about 70% identicalto SEQ ID NO:4, wherein the protein has prelamin A or lamin A biologicalactivity. In this embodiment, the protein is chemically or recombinantlyattached to a therapeutic agent that increases the half-life of theprotein in cardiac or skeletal muscle tissue.

Yet another embodiment of the present invention relates to a carrier fortherapeutic agents for the treatment of cardiac or skeletal muscledisorders. The carrier consists essentially of an isolated fragment ofSEQ ID NO:4 with inter-nuclear transport domain biological activity, ora biologically active homologue thereof.

Another embodiment of the present invention relates to a therapeuticcomposition for promoting myoblast activation and growth or regenerationof cardiac or skeletal muscle. The composition comprises an isolatedpeptide consisting essentially an isolated fragment of SEQ ID NO:4 withinter-nuclear transport domain biological activity or a biologicallyactive homologue thereof, operatively linked to a therapeutic agent forpromoting myoblast activation and growth or regeneration of cardiac orskeletal muscle.

Yet another embodiment of the present invention relates to a recombinantnucleic acid molecule comprising: (a) a nucleic acid sequence encodingan isolated fragment of SEQ ID NO:4 with inter-nuclear transport domainbiological activity or a biologically active homologue thereof,operatively linked to (b) a nucleic acid sequence encoding a protein forthe promotion of myoblast activation and growth or regeneration ofcardiac or skeletal muscle.

Another embodiment of the present invention relates to a method topromote myoblast activation and regeneration of damaged, degenerated oratrophied cardiac and skeletal myocytes. The method includes the step ofadministering to a patient that has damaged, degenerated or atrophiedcardiac or skeletal myocytes an agent selected from: (a) any of theabove-described peptides; (b) any of the above-described compositions;(c) any of the above-described recombinant nucleic acid molecules; and(d) any of the above-described therapeutic proteins.

Yet another embodiment of the present invention relates to a method tostimulate cardiac or skeletal muscle growth in a mammal. The methodincludes the step of administering to a mammal an agent selected from:(a) any of the above-described peptides; (b) any of the above-describedcompositions; (c) any of the above-described recombinant nucleic acidmolecules; and (d) any of the above-described therapeutic proteins.

Another embodiment of the present invention relates to a method to treatcardiac and skeletal muscle disorders. The method includes the step ofadministering to a patient that has a cardiac or skeletal muscledisorder, an agent selected from: (a) any of the above-describedpeptides; (b) any of the above-described compositions; (c) any of theabove-described recombinant nucleic acid molecules; and (d) any of theabove-described therapeutic proteins. Cardiac and skeletal muscledisorders to treat using the method of the present invention include,but are not limited to, dilated cardiomyopathy, Emery-Dreifuss musculardystrophy, limb-girdle muscular dystrophy, partial lipodystrophy, axonalneuropathy, and mandibuloacral dysplasia.

Yet another embodiment of the present invention relates to a method toidentify compounds that regulate myoblast activation anddifferentiation. The method includes the steps of: (a) contacting a cellthat expresses a prelamin A protein or a prelamin A pre peptide with atest compound under conditions suitable for modulation of the activityof the prelamin A protein or prelamin A pre peptide by the testcompound; and (b) detecting modulation of the activity of the prelamin Aprotein or prelamin A pre peptide by the test compound. The step ofdetecting can include, but is not limited to, detecting whether the testprotein regulates prelamin A pre peptide transport in a cell; detectingwhether the test protein regulates the processing of prelamin A in acell; detecting whether the test protein regulates myoblastdifferentiation; detecting binding between the prelamin A protein orprelamin A pre peptide and the test compound (e.g., by a yeast twohybrid assay). In one aspect, the test compound is a protein encoded bya gene that is a candidate for regulation of prelamin A processing orprelamin A pre peptide transport in the cell. For example, the gene canbe a human homologue of a gene in the yeast a-type mating pheromonesignaling pathway, or the gene can be a gene encoding a candidatereceptor for the prelamin A pre peptide. In another aspect, the testcompound is a pharmaceutical compound. In one aspect, the cellexpressing the prelamin A protein or prelamin A pre peptide is adifferentiating cardiac myocyte or a differentiating skeletal myocyte.In another aspect, the cell expressing the prelamin A protein orprelamin A pre peptide has been transfected with a nucleic acid moleculeencoding the prelamin A protein or prelamin A pre peptide. In yetanother aspect, the prelamin A is processing deficient. In yet anotheraspect, the cell is a prelamin A processing deficient cell.

Yet another embodiment of the present invention relates to a method toidentify compounds that regulate myoblast activation anddifferentiation. The method includes the steps of: (a) contacting aprelamin A protein or a prelamin A pre peptide with a test compoundunder conditions suitable for binding of the prelamin A protein orprelamin A pre peptide by the test compound; and (b) detecting bindingof the prelamin A protein or prelamin A pre peptide by the testcompound. Binding can be detected by any suitable technique including,but not limited to, a yeast two hybrid assay or immunoprecipitationassay.

Another embodiment of the present invention relates to a method toidentify compounds that regulate myoblast activation and differentiationin a cell. The method includes the steps of: (a) contacting an isolatedprelamin A processing-deficient cell with a test compound for regulationof myoblast activation and differentiation; and (b) detecting whetherthe test compound regulates an activity in the cell selected from thegroup consisting of: prelamin A processing, prelamin A pre peptidetransport, and myoblast activation or differentiation, as compared to inthe absence of the test compound. In one aspect, the isolated prelamin Aprocessing-deficient cell is selected from: a cell transfected with anucleic acid sequence encoding a processing deficient prelamin A proteinand a prelamin A processing deficient cell that has been isolated from apatient. In one aspect, the cell is transfected with a nucleic acidsequence encoding a prelamin A protein. In another aspect, the cell istransfected with a nucleic acid sequence encoding a processing-deficientprelamin A protein. In another aspect, the processing-deficient prelaminA is a naturally occurring processing-deficient prelamin A protein. Inyet another aspect, the processing-deficient prelamin A is asynthetically created processing-deficient prelamin A protein. Inanother aspect, the cell endogenously expresses a processing-deficientprelamin A protein. In one aspect, the cell is selected from a cardiacmyocyte and a skeletal myocyte. In another aspect, the cell is aprelamin A processing deficient cell that has been isolated from apatient, wherein the cell expresses a prelamin A protein comprising amutation (with respect to SEQ ID NO:4) selected from: Arg60Gly,Leu85Arg, Glu203Gly, Arg89Leu, Asn19Lys, and Arg377His.

In this embodiment of the invention, the step of detecting can include,but is not limited to, detecting whether the test compound increasesprelamin A processing in the cell as compared to in the absence of thecompound; detecting whether the test compound increases prelamin A prepeptide transport in the cell as compared to in the absence of thecompound; detecting whether the test compound increases myoblastactivation or differentiation in the cell as compared to in the absenceof the compound; or detecting an increase in myoblast activation anddifferentiation in the absence of correcting the prelamin A processingdeficiency.

In one aspect of this embodiment of the invention, the test compound isa homologue of prelamin A pre peptide with putative prelamin A prepeptide biological activity. In another aspect, the test compound is apharmaceutical compound with putative prelamin A pre peptide biologicalactivity. In another aspect, the test compound is a homologue ofprelamin A with putative prelamin A biological activity. In anotheraspect, the test compound is a candidate protein for a prelamin Aprocessing enzyme, or a gene encoding the candidate protein. In yetanother aspect, the test compound is a candidate protein for adownstream prelamin A pre peptide signal transduction protein, or a geneencoding the candidate protein. In another aspect, the test compound isa putative pharmaceutical compound for use in the treatment of cardiacand skeletal muscle disorders, wherein an increase in the processing ofprelamin A in the cell or an increase in myoblast activation anddifferentiation in the presence of the compound as compared to in theabsence of the compound indicates that the compound is a therapeuticcompound for use in the treatment of cardiac and skeletal muscledisorders.

Yet another embodiment of the present invention relates to a method toidentify human genes that regulate myoblast activation anddifferentiation. The method includes the steps of: (a) contacting aprobe with a source of human DNA from heart or skeletal muscle tissueunder moderate stringency conditions, wherein the probe is a nucleicacid sequence from a gene in the yeast a-type mating pheromone signaltransduction pathway; (b) identifying genes in the source of human DNAthat hybridize to the probe; and (c) detecting whether genes thathybridize to the probe encode a protein that corrects a prelamin Aprocessing deficiency or that increases myoblast activation anddifferentiation. In one aspect, the gene in the yeast a-type matingpheromone signal transduction pathway is a gene that is associated witha biological function selected from: transcriptional activation ofpheromone responsive genes, post-transcriptional blockade of the cellcycle, and cell fusion pathway activation.

Another embodiment of the present invention relates to a method toidentify an inhibitor of prelamin A farnesylation. The method includesthe steps of: (a) contacting an isolated cell that expresses prelamin Awith a putative regulator of prelamin A farnesylation; and (b) detectingwhether farnesylation of prelamin A is inhibited by the putativeregulator. In one aspect, cell is selected from the group consisting ofa differentiating cardiac myocyte and a differentiating skeletalmyocyte, and in another aspect, the cell has been transfected with anucleic acid molecule encoding prelamin A. The step of detecting caninclude, but is not limited to, detecting whether prelamin Afarnesylation is reduced as compared to in the absence of the putativeinhibitor compound. In one aspect, the method further comprises a step(c) of detecting whether inhibitors of prelamin A farnesylation detectedin step (b) regulate prelamin A processing in the cell, whereindetection of reduced prelamin A processing in the presence of theregulator indicates that the regulator may be useful for treatment ofmuscle cell cancers. In another aspect, the method further includes step(c) of detecting whether inhibitors of prelamin A farnesylation detectedin step (b) cause myoblast dissociation or myoblast cell death, whereindetection of increased myoblast dissociation or myoblast cell death inthe presence of the regulator indicates that the regulator may be usefulfor treatment of muscle cell cancers.

Yet another embodiment of the present invention relates to a method totreat a muscle cell cancer, comprising administering to a patient with amuscle cell cancer or metastatic cancer thereof a compound that inhibitsprelamin A processing and myoblast differentiation and is toxic tomyocytes. The muscle cell cancer can include, but is not limited to,myosarcoma, myeloma, myoma, rhabdomyosarcoma, and malignant uterinefibroids. In one aspect, the compound inhibits farnesylation of prelaminA. In another aspect, the compound is a statin that is toxic tomyocytes. The compound can be identified by the methods described above.

Another embodiment of the invention relates to a processing deficientprelamin A peptide. The processing deficient prelamin A peptide consistsessentially of an amino acid sequence that differs from SEQ ID NO:4 byat least one substitution, deletion or insertion that results in adecrease in a prelamin A or prelamin A pre peptide biological activityselected from: (a) prelamin A processing to release a prelamin A prepeptide consisting of SEQ ID NO:2 or a biologically active homologuethereof; (b) prelamin A pre peptide signal transduction; (c)synchronization of intercellular signaling with changes in lamin Alocalization and nuclear lamina morphology that occur early in myoblastdifferentiation; (d) synchronization of transcriptional regulation ofmuscle-specific genes or cell cycle arrest that occurs concomitant withmyoblast differentiation; (e) formation of normal nuclear laminastructure; and/or (f) induction of myoblast activation anddifferentiation. In one aspect, the processing deficient prelamin Apeptide consists essentially of an amino acid sequence that differs fromSEQ ID NO:4 by a substitution of an amino acid residue in SEQ ID NO:4selected from: Arg60, Leu85, Glu203, Arg89, Asn195, Arg377, Tyr646,G649, N650, P653, R654, P658, Q659, N660, Cys661, S662, I663 and M664.In another aspect, the substitution is selected from: Arg60Gly,Leu85Arg, Glu203Gly, Arg89Leu, Asn19Lys, and Arg377His.

Another embodiment of the invention relates to an isolated celltransfected with a processing deficient prelamin A peptide as describedabove.

BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION

FIG. 1 is a digitized image of an analysis of prelamin A GFP-fusionprotein expression and processing.

FIG. 2 is an alignment of the amino acid sequence of the pre peptideportion of prelamin A from 5 different animal species.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery by the present inventorof the mechanisms by which a class of genetic mutations results in heartdisease and muscular dystrophies. The present invention also relates tothe identification of therapeutic agents useful for treating suchdiseases, as well as for generally promoting myoblast activation and thegrowth or regeneration of cardiac and skeletal muscle. Such agents willalso be useful, for example, in cases where heart and skeletal musclehave been damaged by non-disease pathways and aging. The presentinvention also relates to use of mutant protein and cell lines for thefurther identification of the genes and proteins that are relevant forproper heart and skeletal muscle function, as well as cancerdevelopment. The present invention also relates to the use of variousgenes, proteins, and cell lines described herein to identify additionalgene and protein targets for the treatment of heart and skeletal musclediseases.

More specifically, the present inventor has discovered that variousmutations in the lamin A/C gene that are associated with certain cardiacand skeletal muscle disorders affect prelamin A processing, sub-cellularprelamin A/lamin A localization, and nuclear lamina formation in thecell types affected by these diseases. The inventor has shown thatexpression of prelamin A proteins containing such mutations results inaberrant cardiac and skeletal myocyte differentiation, morphology andorganization, reflecting what is seen in patients with these diseases.This is believed to be the first demonstration of any biologicallysignificant effect of any lamin A/C disease mutations.

The inventor's discovery demonstrates that improper prelamin Aprocessing results in severely aberrant cardiac and skeletal myocytedifferentiation. This is believed to be the first identification of acellular process that is affected by preventing prelamin A processing.These results indicate that the “pre” peptide of prelamin A serves ananalogous function to that of the S. cerevisiae a-type mating factor ora-type mating pheromone, which is the only known protein that isprocessed in a similar manner to prelamin A. The present inventor's dataindicate that the “pre” sequence of prelamin A functions as a signalingmolecule when proteolytically released from the prelamin A protein.Based on the experimental results provided herein, and by the presentinventor's analogy to the demonstrated function of the yeast a-typemating pheromone, the “pre” sequence of prelamin A indicates theproximity and direction of mononucleate myoblasts during differentiationand cell fusion to generate multinucleate myocytes. The removal of the“pre” sequence results in the synchronized incorporation of the maturelamin A protein into the nuclear lamina, altering the lamina structureand affecting gene transcription and/or cell cycle arrest. Consequently,lamin A/C mutations affecting prelamin A processing, “pre” signaling, orlamina formation will result in disease.

Mutations in the human lamin A/C gene cause cardiac and skeletal muscledisease, as well as diseases affecting adipocytes and neurons (seeBackground discussion). As lamin A/C is expressed in nearly all adultcell types, and is directly involved in many nuclear processes, themechanisms by which mutations in this gene lead to tissue-specificphenotypes has led to considerable speculation regarding putativedisease mechanisms. While evidence has been presented demonstrating thatthe lamin A/C mutations resulting in partial lipodystrophy affectinteractions with an adipocyte-specific transcription factor (Lloyd etal., (2002) Hum Mol Genet 11:769-77), no specific functional effects oflamin A/C disease mutations on cardiac or skeletal muscle cell biologyhave been demonstrated. The present inventor has directly demonstratedherein that mutations in the lamin A/C gene associated with dilatedcardiomyopathy affect nuclear lamina structure and prelamin A processingresulting in aberrant myoblast differentiation.

Prelamin A processing proceeds through a sequential series ofpost-translational protein modifications (Sinensky et al., (1994),supra). While a number of studies have demonstrated that the properprocessing and subsequent removal of the prelamin A tail is necessaryfor the efficient incorporation of mature lamin A into the nuclearlamina, the biological significance of prelamin A processing hasremained elusive.

The only protein known to be post-translationally processed in the samemanner as prelamin A is the yeast a-type mating pheromone (yeasta-factor) (Boyartchuk & Rine (1998) Genetics 150:95-101). This matingfactor is expressed as pre-pro-protein, and the farnesylated,carboxymethylated C-terminal peptide that is released by endoproteolyticcleavage is the biologically active species. Yeast a-factor is a peptidepheromone that indicates the proximity and distance of adjacent yeasthaploid cells during the process of yeast mating, which results in cellfusion and the production of diploid yeast cells. The yeast a-factorpeptide binds to a trimeric G-protein coupled receptor, which activatesa mitogen-activated protein (MAP) kinase. The MAP kinase then activatesthree distinct pathways leading to transcriptional activation ofpheromone response regulated genes, post-transcriptional blockade of thecell cycle, and polarized morphogenesis (for review, see Elion, E. A.(2000) Curr Opin Microbiol 3:573-81). As a result of the presentinventor's discovery, it can now be seen that the fact that prelamin Aprocessing is analogous to yeast a-factor processing is supported by thefinding that knockout mice which do not express the murine homologue(Zmpste24) of one of the yeast endoproteases responsible for a-factorprocessing (Ste24) are defective in prelamin A processing, and displayaberrant cardiac and skeletal muscle morphologies that are phenocopiesof those seen in the lamin A/C knockout mouse (Bergo et al., (2002),supra).

The present inventor discloses herein that proper prelamin A processingis necessary for the release of the “pre” peptide of prelamin A, whichfunctions in analogous fashion to yeast a-factor in intercellularsignaling between mononucleated myoblasts during cell fusion and theformation of multinucleated myocytes. Furthermore, as the removal of theprelamin A tail is necessary for the incorporation of mature lamin Ainto the nuclear lamina (Lutz et al., (1992), supra; Izumi et al.,(2000), supra), the present inventor proposes that prelamin A processingsynchronizes intercellular signaling with the changes in lamin Alocalization and nuclear lamina morphology that occur early in myoblastdifferentiation (Chaly et al., (1996) J Cell Biochem 62:76-89;Muralikrishna et al., (2001) J Cell Sci 114:4001-11). These changes inlamina structure are likely to be involved in the transcriptionalregulation of muscle-specific genes and/or the cell cycle arrest thatoccurs concomitant with myoblast differentiation.

The present inventor's model explains the cardiac and skeletalmuscle-specific abnormalities observed in patients with DCM,Emery-Dreifuss muscular dystrophy and limb-girdle muscular dystrophy, aswell as those seen in the lamin A/C and Zmpste24 knockout mice. Inaddition, the finding that prelamin A processing is necessary for thefusion of mononucleated cells into multinucleated myotubes explains whyno biological function could be assigned by prior investigators toprelamin A processing in non-differentiating mononucleated cell lines.Finally, the model described herein indicates that lamin A/C mutationsthat affect prelamin A processing, “pre” signaling, or nuclear laminaformation will result in aberrant myoblast differentiation, andconsequently explains why mutations spread throughout the lamin A/C genecause cardiac and skeletal muscle disease.

Shortly after the discovery that mutations in the human lamin A/C genecause EDMD (Bonne et al., (1999), supra), Brodsky and colleagues(Brodsky et al., (2000), supra) and others (Fatkin et al., (1999),supra), identified disease mutations in families with DCM. Additionalunpublished studies carried out in the present inventor's lab have nowdemonstrated that GFP-prelamin A fusion proteins containing DCM and EDMDdisease mutations resulted in aberrant lamina formation in mononucleatecell lines, as assessed by direct fluorescence microscopy. Similarstudies were reported by others who used mutant lamin proteins taggedwith peptide markers (Ostlund et al., (2001) J Cell Sci 114:4435-45;Raharjo et al., (2001) J Cell Sci 114:4447-57), and who demonstratedthat a protein that interacts with lamin A, emerin, was mislocalized asa result of mutant lamin expression. These studies required the use ofindirect immunofluorescence microscopy. Due to the use of thistechnique, the lamina morphology results reported were ambiguous as theauthors could not demonstrate if the lamina morphologies observed weredue to the actual structures the mutant proteins formed, or justreflected changes in antibody-protein interactions brought about by themutations. Previous reports (Chaly et al., (1996), supra; Muralikrishnaet al., (2001), supra) had shown that antibody access could be inhibitedby changes in lamina structure. In addition, the present inventor hasdata (currently unpublished) that directly demonstrates that indirectimmunofluorescence studies of these very same mutations result inmisrepresentation of the true lamina structure.

Recent screening of additional families with DCM by the presentinventor's research group identified two additional mutations (Taylor etal., 2002, supra) responsible for this disease. The present inventorrecently demonstrated that one of these two mutations was not processedcorrectly, and that expression of 4 of the 6 DCM mutations being studiedresulted in aberrant cardiac and skeletal myoblast differentiation, asevidenced by misshapen and disorganized multinucleate myocytes. Themutation which prevents prelamin A processing produces the most severephenotype. As discussed above, the present inventor's discovery isbelieved to be the first identification of a cellular process affectedby any lamin A/C disease mutation. This is also believed to be the firstdemonstration of a specific biological process affected by preventingprelamin A processing.

Prior to the present invention, there were no published data identifyingthe cellular function of prelamin A processing, or the mechanism bywhich mutations in the lamin A/C gene, or the deletion of the lamin A/Cgene and enzymes that process prelamin A, lead to cardiac and skeletalmuscle abnormalities. The present inventor's findings are completelyunique, and show that prelamin A processing is necessary for thebiological process whereby mononucleate myoblasts differentiate and fuseto form multinucleate myocytes. The present inventor's data is also thefirst to demonstrate that lamin A/C disease mutations can interfere withthis same process. This is the first demonstration of a functional celldefect arising from any lamin A/C disease mutations. The finding by thepresent inventor that a prelamin A processing mutant causes aberrantmyocyte differentiation was the key factor in elucidating that the “pre”sequence of prelamin A functions in an analogous manner to yeast a-typemating pheromone, which signals and synchronizes haploid yeast cellsprior to fusion to become a diploid cell.

While many disease mutations have been identified and shown to producedisease phenotypes in transgenic and/or knock-out mouse models, themechanism by which these mutations exert their effects have rarely beenidentified. Without an understanding of how the mutations exert theireffects, it is not possible to efficiently design therapeutic strategiesto treat the disease. The identification of the cellular processeffected by lamin A/C disease mutations and prevention of prelamin Aprocessing by the present inventor represents an exponential leap in theunderstanding of these disease processes, as well as in the ability todesign therapies to prevent these and other cardiac and skeletal musclediseases.

Prior to the present invention, investigators had failed to discover thelink between prevention of prelamin A processing and myoblastactivation, including differentiation and cell fusion, which may beexplained by a variety of reasons. First, the lamins have multipleactivities. Lamin A provides structural support for the nucleus, bindstranscription factors, interacts with RNA processing factors, isdynamically involved in cell cycle regulation, binds chromatin andhistone proteins, and is involved in apoptosis. Consequently, manydifferent models have been proposed to explain why lamin A/C mutationscause heart and muscle disease. The most generally accepted modelsuggested that structural perturbations to the lamina are exacerbated bycardiac and skeletal muscle contraction, causing the nuclei in thesetissues to be damaged.

Second, the farnesylation of all other human proteins serves to anchorthese proteins to membranes. Consequently, the finding by priorinvestigators that removal of the farnesylated pre-peptide of prelamin Awas necessary for the incorporation of the mature lamin A protein intothe lamina led most to believe that the “pre” sequence functions toregulate the production and activity of mature lamin A, and has noactivity of its own. This concept was likely reinforced by the fact thatthe peptide from which yeast a-type mating pheromone is released has nobiological activity. As a result, the fact that lamin A had clearbiological activities apparently led most investigators to believe that,in contrast to yeast a-type mating pheromone, the “pre” sequence was nota biologically active part of the prelamin A protein.

While the processing of prelamin A does allow the mature lamin A proteinto incorporate into the nuclear lamina, thereby synchronizing changes inlamina structure associated with myocyte differentiation with therelease of the “pre” peptide signal, the nearly ubiquitous expressionpattern of the lamin A/C gene prevented the identification of the celltypes in which this processing takes place. In other words, becauseresearchers didn't know the function of prelamin A processing, theydidn't know what cell types to use as models. Without using the correctcell types (e.g., differentiating cardiac and skeletal myocytes) therewas no way to identify the biological function of prelamin A processingas the present inventor has now done.

Furthermore, the fact that other disease-causing mutations in the laminA/C gene, as well as a knock-out of the mouse lamin A/C gene, were shownto also cause adipocyte and neuronal abnormalities complicated theelucidation of the function of prelamin A by demonstrating that musclewas not the only tissue type affected by mutations in lamin A/C.Furthermore, muscle tissue has many other unique characteristics inaddition to containing multinucleated cells derived from the fusion ofmononucleated myoblasts.

Finally, extensive studies of prelamin A processing have been performedfor more than ten years, using both chemical agents and laboratoryinduced mutations, without identifying the biological function of thispost-translational modification. These studies failed to identify thefunction of prelamin A processing primarily because they utilizedmononucleated cell lines in which prelamin A processing has nobiological function. One key factor in elucidating the function ofprelamin A processing by the present inventor was the determination inthe present inventor's laboratory that a DCM mutation prevented properprocessing and resulted in aberrant cardiac and skeletal myocytedifferentiation. Furthermore, it was the identification of the functionof prelamin A processing combined with the findings that all of thedisease mutations studied affected lamina structure, and 4 of the 6mutations caused aberrant myocyte differentiation, that lead to thediscovery that all of the disease mutations were affecting the samecellular process. Once this realization was made, the present inventorwas able to re-evaluate an extensive amount of data produced in hislaboratory and published in the literature, and discovered thatmutations that caused aberrant lamina formation and which preventedprelamin A processing were effecting two halves of a single signalingpathway which mediates myocyte differentiation and fusion. Theconcomitant identification in the present inventor's laboratory ofchanges in the lamina architecture, altered prelamin A processing, andaberrant cardiac and skeletal myocyte differentiation resulting fromlamin A/C disease mutations, identification of the mechanism by whichlamin A/C mutations lead to cardiac and skeletal muscle disease, and thefunction of prelamin A processing, led to the present invention and theproducts and methods described herein.

The discovery by the present inventor has led to various aspects of thepresent invention, including, but not limited to, the provision of:isolated peptides encoding the prelamin pre peptide, the prelamin Aprotein, homologues, mimetics, and fragments thereof, and nucleic acidmolecules encoding the same, as therapeutic molecules or compositionsfor the promotion of myoblast activation and differentiation and for thetreatment of cardiac and skeletal muscle disorders; methods to identifycompounds useful for the regulation of prelamin A processing andmyoblast activation and differentiation; methods to identify genes andproteins in the prelamin A processing pathway and prelamin A pre peptidesignal transduction pathway; methods to promote myoblast activation anddifferentiation and to treat cardiac and skeletal muscle disorders;methods to identify compounds for the treatment of muscle cell cancersand the use of such compounds in therapeutic methods; and prelamin Aprocessing-deficient proteins and cell lines.

According to the present invention, prelamin A is a pre-proteinexpression product of the lamin A/C gene that is post-translationallyprocessed to yield (1) lamin A and (2) the “pre” peptide. The nucleotidesequence of the cDNA encoding human prelamin C (Database Accession No.NM_(—)005572) is represented herein by SEQ ID NO:7. The cDNA nucleicacid sequence encoding human prelamin A (Database Accession No.NM_(—)170707) is represented here by SEQ ID NO:3. SEQ ID NO:3 encodesthe human prelamin A protein that has an amino acid sequence representedherein by SEQ ID NO:4. The nucleic acid and amino acid sequence ofprelamin A is also known for a variety of other animal species,including, but not limited to: mouse, chicken, Xenopus laevis (Africanclawed frog), and Danio rerio (zebra fish). The nucleic acid sequence ofmouse prelamin A (Database Accession No. BC015302) is represented hereinby SEQ ID NO:8. SEQ ID NO:8 encodes the mouse prelamin A protein thathas an amino acid sequence represented by SEQ ID NO:9. The nucleic acidsequence of chicken prelamin A (Database Accession No. X16879) isrepresented herein by SEQ ID NO:10. SEQ ID NO:10 encodes the chickenprelamin A protein that has an amino acid sequence represented by SEQ IDNO:11. The nucleic acid sequence of Xenopus laevis prelamin A (DatabaseAccession No. X06345) is represented herein by SEQ ID NO:12. SEQ IDNO:12 encodes the Xenopus laevis prelamin A protein that has an aminoacid sequence represented by SEQ ID NO:13. The nucleic acid sequence ofDanio rerio prelamin A (Database Accession No. AF397016) is representedherein by SEQ ID NO:14. SEQ ID NO:14 encodes the Danio rerio prelamin Aprotein that has an amino acid sequence represented by SEQ ID NO:15.

Prelamin A processing proceeds through a sequential series ofpost-translational protein modifications (Sinensky et al., (1994),supra). The cysteine residue in the prelamin A C-terminal CAAX motif(C=Cysteine, A=aliphatic amino acid, X=any amino acid) (e.g., positions661-664 of SEQ ID NO:4) is farnesylated, followed by the endoproteolyticremoval of the C-terminal tripeptide (−AAX). The now C-terminal cysteineresidue is carboxymethylated, and finally the C-terminal 15 amino acidpeptide (in humans) (i.e., the “pre” peptide) containing the modifiedcysteine residue (e.g., positions 647-661 of SEQ ID NO:4) is removed byan additional endoproteolytic processing step. The nucleic acid sequenceof human lamin A is represented herein by SEQ ID NO:5. SEQ ID NO:5encodes the lamin A protein having the amino acid sequence representedby SEQ ID NO:6.

The nucleic acid sequence of the processed “pre” peptide from humanprelamin A is represented herein by SEQ ID NO:1. SEQ ID NO:1 encodes a15 amino acid peptide (referred to herein as “pre”, “pre peptide”, or“prelamin A pre peptide”) having an amino acid sequence representedherein by SEQ ID NO:2. One of skill in the art will know, based on thesequence of the prelamin A proteins from other animal species and theknowledge of how the protein is processed, the sequence of the processedlamin A and “pre” peptides corresponding to these other prelamin Aproteins. For example, the mouse pre peptide is, by homology to thehuman pre peptide: LLGNSSPRSQSSQNC (SEQ ID NO:16). The chicken prepeptide has been shown to be: VLGGAGPRRQAPAPQGC (SEQ ID NO:17). The prepeptide for Xenopus laevis is, by homology to the human pre peptide:IVGNGQRAQVAPQNC (SEQ ID NO:18). The pre peptide for Danio rerio is, byhomology to the human pre peptide: IVSNDKPRQAGPKVDNC (SEQ ID NO:19). Thesequences of the lamin A and “pre” peptides for any known prelamin Aprotein or nucleic acid sequence encoding the same are explicitlyencompassed by the present invention. The complete sequences representedby each of the sequence database accession numbers recited herein areincorporated herein by reference. An alignment of the prelamin A “pre”peptide amino acid sequences (including the entire CAAX motif that isultimately processed to reveal a modified cysteine C terminus), is shownin FIG. 2.

Although the embodiments of the invention are discussed below withregard to the human prelamin A and prelamin A pre peptide sequences(e.g., SEQ ID NO:4 and SEQ ID NO:2, respectively), it is to beunderstood that the present invention expressly encompasses thesubstitution of sequences of prelamin A or prelamin A pre peptide fromany other animal species (including from mouse, chicken, Xenopus laevisor Danio rerio discussed above) in any of the embodiments describedbelow.

One embodiment of the present invention relates to an isolated peptideselected from: (a) a peptide consisting essentially of SEQ ID NO:2; (b)a biologically active fragment of SEQ ID NO:2; (c) a peptide consistingessentially of an amino acid sequence that is at least about 70%identical to SEQ ID NO:2 with the biological activity of SEQ ID NO:2;and/or (d) a peptide consisting essentially of an amino acid sequencethat differs from SEQ ID NO:2 by at least one substitution, deletion orinsertion of an amino acid residue at a position of SEQ ID NO:2 selectedfrom the group consisting of: 1, 2, 5, 6, 9, 10, 11, 12, 13 and/or 14,wherein the peptide has the biological activity of SEQ ID NO:2. Asdiscussed above, SEQ ID NO:2 represents the amino acid sequence of aprelamin A pre peptide.

Another embodiment of the present invention relates to an isolatedpeptide selected from: (a) a protein comprising an amino acid sequencerepresented by SEQ ID NO:4; (b) a protein comprising biologically activefragment of SEQ ID NO:4; and (c) a protein comprising an amino acidsequence that is at least about 70% identical to SEQ ID NO:4, whereinthe protein has prelamin A or lamin A biological activity. In oneaspect, this protein is chemically or recombinantly attached to atherapeutic agent that increases the half-life of the protein in cardiacor skeletal muscle tissue. SEQ ID NO:4 represents the amino acidsequence of a prelamin A protein of the invention.

Yet another embodiment of the present invention relates to an isolatedpeptide that consists essentially of an isolated fragment of SEQ ID NO:4with inter-nuclear transport domain biological activity, or abiologically active homologue thereof. This fragment would beparticularly useful as a carrier (e.g., as a fusion partner or carrierto be linked to a compound) for therapeutic agents for the treatment ofcardiac or skeletal muscle disorders. A “carrier” refers to anysubstance or vehicle suitable for delivering a therapeutic compositionuseful in a therapeutic method (described below) to a suitable in vivoor ex vivo site. Methods of conjugating or operatively linking theabove-described protein or fragment to another protein or to anon-protein compound are well known in the art.

The “pre” peptide of prelamin A is a small, 15 amino acid, naturallyoccurring, easily synthesized, signaling peptide that specificallypromotes proper cardiac and skeletal myoblast fusion, myocytedifferentiation, and myocyte organization in adults. Consequently, thispeptide is an excellent drug candidate as it will specifically promotecell fusion and regeneration of cardiac and skeletal myocytes damaged bydisease or other factors. The peptide could be given in its proteinform, or introduced as a cDNA by gene therapy. The prelamin A cDNA isalso an excellent candidate for gene therapy of cardiac and skeletalmuscle disorders and degeneration (or the protein encoded thereby couldbe delivered). The present inventor's data shows that this protein israpidly transferred between the multiple nuclei within a myocyte, andaffects the morphology and organization of the transfected myocytes aswell as that of adjacent untransfected myocytes. Consequently, theprelamin A cDNA would be a highly potent and efficacious gene therapytreatment.

According to the present invention, an isolated protein or peptide, suchas a prelamin A protein or pre peptide, is a protein (including apolypeptide or peptide) that has been removed from its natural milieu(i.e., that has been subject to human manipulation) and can includepurified proteins, partially purified proteins, recombinantly producedproteins, and synthetically produced proteins, for example. As such,“isolated” does not reflect the extent to which the protein has beenpurified. Preferably, an isolated protein such as a prelamin A proteinof the present invention is produced recombinantly. An isolated peptide,such as the pre peptide, can be produced synthetically (e.g.,chemically, such as by peptide synthesis) or recombinantly. In addition,and by way of example, a “human prelamin A protein” refers to a prelaminA protein (generally including a homologue of a naturally occurringprelamin A protein) from a human (Homo sapiens), or to a prelamin Aprotein that has been otherwise produced from the knowledge of thestructure (e.g., sequence), and perhaps the function, of a naturallyoccurring prelamin A protein from Homo sapiens. In other words, generalreference to a human prelamin A protein includes any prelamin A proteinthat has substantially similar structure and function of a naturallyoccurring prelamin A protein from Homo sapiens or that is a biologicallyactive (i.e., has biological activity) homologue of a naturallyoccurring prelamin A protein from Homo sapiens as described in detailherein. As such, a human prelamin A protein can include purified,partially purified, recombinant, mutated/modified and syntheticproteins. The same description applies to reference to other proteins orpeptides described herein, such as the pre peptide of prelamin A.

According to the present invention, the terms “modification” and“mutation” can be used interchangeably, particularly with regard to themodifications/mutations to the primary amino acid sequences of prelaminA or pre (or nucleic acid sequences) described herein. The term“modification” can also be used to describe post-translationalmodifications to a protein or peptide including, but not limited to,methylation, farnesylation, carboxymethylation, geranyl geranylation,glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, and/or amidation. Modifications can alsoinclude, for example, complexing a protein or peptide with a lipidcarrier. Such modifications can be considered to be mutations if themodification is different than the post-translational modification thatoccurs in the natural, wild-type protein or peptide.

As used herein, the term “homologue” is used to refer to a protein orpeptide which differs from a naturally occurring protein or peptide(i.e., the “prototype” or “wild-type” protein) by one or more minormodifications or mutations to the naturally occurring protein orpeptide, but which maintains the overall basic protein and side chainstructure of the naturally occurring form (i.e., such that the homologueis identifiable as being related to the wild-type protein). Such changesinclude, but are not limited to: changes in one or a few amino acid sidechains; changes one or a few amino acids, including deletions (e.g., atruncated version of the protein or peptide) insertions and/orsubstitutions; changes in stereochemistry of one or a few atoms; and/orminor derivatizations, including but not limited to: methylation,farnesylation, geranyl geranylation, glycosylation, carboxymethylation,phosphorylation, acetylation, myristoylation, prenylation, palmitation,and/or amidation. A homologue can have either enhanced, decreased, orsubstantially similar properties as compared to the naturally occurringprotein or peptide. A homologue can include an agonist of a protein orpeptide or an antagonist of a protein or peptide.

Homologues can be the result of natural allelic variation or naturalmutation. A naturally occurring allelic variant of a nucleic acidencoding a protein is a gene that occurs at essentially the same locus(or loci) in the genome as the gene which encodes such protein, butwhich, due to natural variations caused by, for example, mutation orrecombination, has a similar but not identical sequence. Allelicvariants typically encode proteins having similar activity to that ofthe protein encoded by the gene to which they are being compared. Oneclass of allelic variants can encode the same protein but have differentnucleic acid sequences due to the degeneracy of the genetic code.Allelic variants can also comprise alterations in the 5′ or 3′untranslated regions of the gene (e.g., in regulatory control regions).Allelic variants are well known to those skilled in the art.

Homologues can be produced using techniques known in the art for theproduction of proteins including, but not limited to, directmodifications to the isolated, naturally occurring protein, directprotein synthesis, or modifications to the nucleic acid sequenceencoding the protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis.

Modifications in protein homologues, as compared to the wild-typeprotein, either agonize, antagonize, or do not substantially change, thebasic biological activity of the homologue as compared to the naturallyoccurring (wild-type) protein. In general, the biological activity orbiological action of a protein refers to any function(s) exhibited orperformed by the protein that is ascribed to the naturally occurringform of the protein as measured or observed in vivo (i.e., in thenatural physiological environment of the protein) or in vitro (i.e.,under laboratory conditions). Modifications of a protein, such as in ahomologue or mimetic (discussed below), may result in proteins havingthe same biological activity as the naturally occurring protein, or inproteins having decreased or increased biological activity as comparedto the naturally occurring protein. Modifications which result in adecrease in protein expression or a decrease in the activity of theprotein, can be referred to as inactivation (complete or partial),down-regulation, or decreased action (or activity) of a protein.Similarly, modifications which result in an increase in proteinexpression or an increase in the activity of the protein, can bereferred to as amplification, overproduction, activation, enhancement,up-regulation or increased action (or activity) of a protein. It isnoted that general reference to a homologue having the biologicalactivity of the wild-type protein does not necessarily mean that thehomologue has identical biological activity as the wild-type protein,particularly with regard to the level of biological activity. Rather, ahomologue can perform the same biological activity as the wild-typeprotein, but at a reduced or increased level of activity as compared tothe wild-type protein.

According to the present invention, an isolated prelamin A protein or anisolated pre peptide (or other isolated protein described herein),including a biologically active homologue or fragment thereof, has atleast one characteristic of biological activity of activity thewild-type, or naturally occurring protein (which can vary depending onwhether the homologue or fragment is an agonist, antagonist, or mimic ofthe wild-type protein). The biological activity of prelamin A caninclude any activity of the pre peptide or of the lamin peptide,including, but not limited to: expression of prelamin A or pre peptide;processing of prelamin A to release the pre peptide and lamin; prepeptide signal transduction, synchronization of intercellular signalingwith changes in lamin A localization and nuclear lamina morphology thatoccur early in myoblast differentiation, synchronization oftranscriptional regulation of muscle-specific genes or cell cycle arrestthat occurs concomitant with myoblast differentiation, induction ofmyoblast activation and differentiation, and incorporation of lamin Ainto the nuclear lamina structure.

Methods of detecting and measuring prelamin A or pre peptide biologicalactivity (which can be applied appropriately to measure agonist orantagonist activity) include, but are not limited to, measurement oftranscription of prelamin A, measurement of translation of prelamin A,measurement of posttranslational modification of prelamin A, measurementof processing of the pre peptide, measurement of pre peptide signaltransduction, measurement of lamin A incorporation into the nuclearlamina structure, measurement of transcriptional regulation ofmuscle-specific genes and/or cell cycle arrest, measurement of nuclearlamina morphology, measurement of pre peptide transport, measurement oflamin A localization, measurement of myocyte cell fusion, and/ormeasurement of myoblast activation and differentiation. It is noted thatan isolated protein of the present invention (including homologues) isnot necessarily required to have the biological activity of thewild-type protein. For example, a prelamin A protein can be a truncated,mutated or inactive protein, for example. Such proteins are useful indiagnostic assays or some screening assays, for example, or for otherpurposes such as antibody production. In a preferred embodiment, theisolated proteins of the present invention (e.g., prelamin A or prepeptide) have biological activity that is similar to that of thewild-type protein (although not necessarily equivalent, as discussedabove).

Methods to measure protein expression levels of this invention include,but are not limited to: Western blot, immunoblot, enzyme-linkedimmunosorbant assay (ELISA), radioimmunoassay (RIA),immunoprecipitation, surface plasmon resonance, chemiluminescence,fluorescent polarization, phosphorescence, immunohistochemical analysis,matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)mass spectrometry, microcytometry, microarray, microscopy, fluorescenceactivated cell sorting (FACS), and flow cytometry, as well as assaysbased on a property of the protein including but not limited to DNAbinding, ligand binding, or interaction with other protein partners.Binding assays are also well known in the art. For example, a BIAcoremachine can be used to determine the binding constant of a complexbetween two proteins. The dissociation constant for the complex can bedetermined by monitoring changes in the refractive index with respect totime as buffer is passed over the chip (O'Shannessy et al. Anal.Biochem. 212:457-468 (1993); Schuster et al., Nature 365:343-347(1993)). Other suitable assays for measuring the binding of one proteinto another include, for example, immunoassays such as enzyme linkedimmunoabsorbent assays (ELISA) and radioimmunoassays (RIA); ordetermination of binding by monitoring the change in the spectroscopicor optical properties of the proteins through fluorescence, UVabsorption, circular dichrosim, or nuclear magnetic resonance (NMR). Toevaluate whether two proteins interact, two hybrid assays (e.g., yeasttwo hybrid assays) are useful and are particularly useful foridentifying proteins (gene products) that interact with prelamin A orpre peptide.

As used herein, an “agonist” of a protein or peptide of the inventionrefers to any compound that is characterized by the ability to agonize(e.g., stimulate, induce, increase, enhance, or mimic) the biologicalactivity of the naturally occurring (wild-type) protein as describedherein. More particularly, an agonist can include, but is not limitedto, a protein, peptide, or nucleic acid that stimulates, induces, mimicsor enhances the activity of the natural ligand, (e.g., prelamin A or prepeptide), and includes homologue of the wild-type protein, a bindingprotein (e.g., an antibody), or any suitable product ofdrug/compound/peptide design or selection which is characterized by itsability to agonize (e.g., stimulate, induce, increase, enhance) thebiological activity of a naturally occurring protein. Agonists can beuseful in methods for regulating myoblast activation and/or the growthor regeneration of cardiac or skeletal muscle.

The phrase, “antagonist” refers to any compound which inhibits (e.g.,antagonizes, reduces, decreases, blocks, reverses, or alters) the effectof a naturally occurring or wild-type protein of the invention or of anagonist thereof, as described above. More particularly, an antagonist iscapable of associating with proteins or other compounds in a mannersimilar to the wild-type protein, or otherwise acts in a manner relativeto the activity of the wild-type protein, such that the biologicalactivity of the wild-type protein is decreased or blocked in a mannerthat is antagonistic (e.g., against, a reversal of, contrary to) to thenatural action of wild-type protein. Such antagonists can include, butare not limited to, a protein, peptide, or nucleic acid (includingribozymes and antisense) or product of drug/compound/peptide design orselection that provides the antagonistic effect. Antagonists can beuseful, for example, in methods for treatment of muscle cell cancers andmetastatic cancers thereof.

According to the present invention, a ribozyme typically containsstretches of complementary RNA bases that can base-pair with a targetRNA ligand, including the RNA molecule itself, giving rise to an activesite of defined structure that can cleave the bound RNA molecule (SeeMaulik et al., 1997, supra). Therefore, a ribozyme can serve as atargeting delivery vehicle for a nucleic acid molecule, oralternatively, the ribozyme can target and bind to RNA encoding prelaminA, for example, and thereby effectively inhibit the translation ofprelamin A.

As used herein, an anti-sense nucleic acid molecule is defined as anisolated nucleic acid molecule that reduces expression of a protein byhybridizing under high stringency conditions to a gene encoding theprotein (including to regulatory regions of the gene encoding theprotein). Such a nucleic acid molecule is sufficiently similar to thenucleic acid sequence encoding the protein that the molecule is capableof hybridizing under high stringency conditions to the coding strand ofthe gene or RNA encoding the natural protein. In a particularlypreferred embodiment, an anti-sense nucleic acid molecule of the presentinvention is the exact complement of the regulatory region or the codingregion of the protein. It is noted that the anti-sense of the codingregion does not necessarily include the anti-sense of the stop codon.

Homologues of prelamin A or pre peptide, including peptide andnon-peptide agonists and antagonists of prelamin A or pre peptide, canbe products of drug design or selection and can be produced usingvarious methods known in the art. Such homologues can be referred to asmimetics. A mimetic refers to any peptide or non-peptide compound thatis able to mimic the biological action of a naturally occurring peptide,often because the mimetic has a basic structure that mimics the basicstructure of the naturally occurring peptide and/or has the salientbiological properties of the naturally occurring peptide. Mimetics caninclude, but are not limited to: peptides that have substantialmodifications from the prototype such as no side chain similarity withthe naturally occurring peptide (such modifications, for example, maydecrease its susceptibility to degradation); anti-idiotypic and/orcatalytic antibodies, or fragments thereof; non-proteinaceous portionsof an isolated protein (e.g., carbohydrate structures); or synthetic ornatural organic molecules, including nucleic acids and drugs identifiedthrough combinatorial chemistry, for example. Such mimetics can bedesigned, selected and/or otherwise identified using a variety ofmethods known in the art. Various methods of drug design, useful todesign or select mimetics or other therapeutic compounds useful in thepresent invention are disclosed in Maulik et al., 1997, MolecularBiotechnology: Therapeutic Applications and Strategies, Wiley-Liss,Inc., which is incorporated herein by reference in its entirety.

A mimetic can be obtained, for example, from molecular diversitystrategies (a combination of related strategies allowing the rapidconstruction of large, chemically diverse molecule libraries), librariesof natural or synthetic compounds, in particular from chemical orcombinatorial libraries (i.e., libraries of compounds that differ insequence or size but that have the similar building blocks) or byrational, directed or random drug design. See for example, Maulik etal., supra.

In a molecular diversity strategy, large compound libraries aresynthesized, for example, from peptides, oligonucleotides, carbohydratesand/or synthetic organic molecules, using biological, enzymatic and/orchemical approaches. The critical parameters in developing a moleculardiversity strategy include subunit diversity, molecular size, andlibrary diversity. The general goal of screening such libraries is toutilize sequential application of combinatorial selection to obtainhigh-affinity ligands for a desired target, and then to optimize thelead molecules by either random or directed design strategies. Methodsof molecular diversity are described in detail in Maulik, et al., ibid.

Maulik et al. also disclose, for example, methods of directed design, inwhich the user directs the process of creating novel molecules from afragment library of appropriately selected fragments; random design, inwhich the user uses a genetic or other algorithm to randomly mutatefragments and their combinations while simultaneously applying aselection criterion to evaluate the fitness of candidate ligands; and agrid-based approach in which the user calculates the interaction energybetween three dimensional receptor structures and small fragment probes,followed by linking together of favorable probe sites.

In one embodiment of the present invention, a prelamin A protein has anamino acid sequence that comprises, consists essentially of, or consistsof, SEQ ID NO:4. SEQ ID NO:4 represents a human prelamin A protein(encoded by nucleic acid sequence SEQ ID NO:3). The present inventionalso includes homologues of SEQ ID NO:4, or fragments of SEQ ID NO:4,wherein the homologue or fragment has prelamin A biological activity(including agonist or antagonist activity), as described previouslyherein.

In one embodiment of the present invention, a pre peptide of prelamin Ahas an amino acid sequence that comprises, consists essentially of, orconsists of, SEQ ID NO:2. SEQ ID NO:2 represents a human pre peptide(encoded by SEQ ID NO:1). The present invention also includes homologuesof SEQ ID NO:2 or fragments of SEQ ID NO:2, wherein the homologue orfragment has pre peptide biological activity (including agonist orantagonist activity), as described previously herein and as described inmore detail below.

In one embodiment, a pre peptide or a prelamin A protein of the presentinvention, including a homologue thereof, has an amino acid sequencethat is at least about 50% identical to an amino acid sequence of SEQ IDNO:2 or SEQ ID NO:4, respectively, over the full length of any of suchsequences, wherein the protein has pre peptide or prelamin A biologicalactivity (which can include agonist or antagonist activity),respectively. In another embodiment, a pre peptide or a prelamin Aprotein useful in the present invention has an amino acid sequence thatis at least about 55% identical, or at least about 60% identical, or atleast about 65% identical, or at least about 70% identical, or at leastabout 75% identical, or at least about 80% identical, or at least about85% identical, or at least about 90% identical, or at least about 95%identical, or at least about 96% identical, or at least about 97%identical, or at least about 98% identical, or at least about 99%identical to SEQ ID NO:2 or SEQ ID NO:4, respectively, over the fulllength of any of such sequences.

In one embodiment of the present invention, a homologue of a protein,such as a prelamin A protein or a prelamin A pre peptide according tothe present invention has an amino acid sequence that is less than about100% identical to the wild-type sequence (e.g., SEQ ID NO:4 or SEQ IDNO:2). In another aspect of the invention, a homologue according to thepresent invention has an amino acid sequence that is less than about 99%identical to the wild-type amino acid sequence, and in anotherembodiment, is less than is less than 98% identical to the wild-typeamino acid sequence, and in another embodiment, is less than 97%identical to the wild-type amino acid sequence, and in anotherembodiment, is less than 96% identical to the wild-type amino acidsequence, and in another embodiment, is less than 95% identical to thewild-type amino acid sequence, and in another embodiment, is less than94% identical to the wild-type amino acid sequence, and in anotherembodiment, is less than 93% identical to the wild-type amino acidsequence, and in another embodiment, is less than 92% identical to thewild-type amino acid sequence, and in another embodiment, is less than91% identical to the wild-type amino acid sequence, and in anotherembodiment, is less than 90% identical to the wild-type amino acidsequence, and so on, in increments of whole integers.

As used herein, unless otherwise specified, reference to a percent (%)identity refers to an evaluation of homology which is performed using:(1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acidsearches, blastn for nucleic acid searches, and blastX for nucleic acidsearches and searches of translated amino acids in all 6 open readingframes, all with standard default parameters, wherein the query sequenceis filtered for low complexity regions by default (described inAltschul, S. F., Madden, T. L., Schääffer, A. A., Zhang, J., Zhang, Z.,Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs.” Nucleic Acids Res.25:3389-3402, incorporated herein by reference in its entirety); (2) aBLAST 2 alignment (using the parameters described below); (3) and/orPSI-BLAST with the standard default parameters (Position-SpecificIterated BLAST). It is noted that due to some differences in thestandard parameters between BLAST 2.0 Basic BLAST and BLAST 2, twospecific sequences might be recognized as having significant homologyusing the BLAST 2 program, whereas a search performed in BLAST 2.0 BasicBLAST using one of the sequences as the query sequence may not identifythe second sequence in the top matches. In addition, PSI-BLAST providesan automated, easy-to-use version of a “profile” search, which is asensitive way to look for sequence homologues. The program firstperforms a gapped BLAST database search. The PSI-BLAST program uses theinformation from any significant alignments returned to construct aposition-specific score matrix, which replaces the query sequence forthe next round of database searching. Therefore, it is to be understoodthat percent identity can be determined by using any one of theseprograms.

Two specific sequences can be aligned to one another using BLAST 2sequence as described in Tatusova and Madden, (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250, incorporated herein by reference inits entirety. BLAST 2 sequence alignment is performed in blastp orblastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search(BLAST 2.0) between the two sequences allowing for the introduction ofgaps (deletions and insertions) in the resulting alignment. For purposesof clarity herein, a BLAST 2 sequence alignment is performed using thestandard default parameters as follows.

For blastn, using 0 BLOSUM62 matrix:

Reward for match=1

Penalty for mismatch=−2

Open gap (5) and extension gap (2) penalties

gap x_dropoff (50) expect (10) word size (11) filter (on)

For blastp, using 0 BLOSUM62 matrix:

Open gap (11) and extension gap (1) penalties

gap x_dropoff (50) expect (10) word size (3) filter (on).

According to the present invention, the term “contiguous” or“consecutive”, with regard to nucleic acid or amino acid sequencesdescribed herein, means to be connected in an unbroken sequence. Forexample, for a first sequence to comprise 30 contiguous (or consecutive)amino acids of a second sequence, means that the first sequence includesan unbroken sequence of 30 amino acid residues that is 100% identical toan unbroken sequence of 30 amino acid residues in the second sequence.Similarly, for a first sequence to have “100% identity” with a secondsequence means that the first sequence exactly matches the secondsequence with no gaps between nucleotides or amino acids.

In another embodiment, a pre peptide homologue or a prelamin A homologueincludes a protein having an amino acid sequence that is sufficientlysimilar to a naturally occurring pre peptide or prelamin A amino acidsequence, respectively, that a nucleic acid sequence encoding thehomologue is capable of hybridizing under moderate, high, or very highstringency conditions (described below) to (i.e., with) a nucleic acidmolecule encoding the naturally occurring protein (i.e., to thecomplement of the nucleic acid strand encoding the naturally occurringamino acid sequence). Preferably, a protein useful in the invention,including a homologue, is encoded by a nucleic acid sequence thathybridizes under moderate, high or very high stringency conditions tothe complement of a nucleic acid sequence that encodes a proteincomprising an amino acid sequence represented by SEQ ID NO:2 or SEQ IDNO:4. Even more preferably, a protein useful in the present invention,including a homologue, is encoded by a nucleic acid sequence thathybridizes under moderate, high or very high stringency conditions tothe complement of the coding region of a nucleic acid sequence selectedfrom SEQ ID NO:1 or SEQ ID NO:3, or fragments thereof. Suchhybridization conditions are described in detail below. A nucleic acidsequence complement of nucleic acid sequence encoding a protein usefulin the present invention refers to the nucleic acid sequence of thenucleic acid strand that is complementary to the strand which encodesthe protein. It will be appreciated that a double stranded DNA whichencodes a given amino acid sequence comprises a single strand DNA andits complementary strand having a sequence that is a complement to thesingle strand DNA. As such, nucleic acid molecules of the presentinvention can be either double-stranded or single-stranded, and includethose nucleic acid molecules that form stable hybrids under stringenthybridization conditions with a nucleic acid sequence that encodes anamino acid sequence of pre peptide or prelamin A, for example, and/orwith the complement of the nucleic acid sequence that encodes any ofsuch amino acid sequences. Methods to deduce a complementary sequenceare known to those skilled in the art. It should be noted that sinceamino acid sequencing and nucleic acid sequencing technologies are notentirely error-free, the sequences presented herein, at best, representapparent sequences of pre peptide and prelamin A of the presentinvention.

As used herein, reference to hybridization conditions refers to standardhybridization conditions under which nucleic acid molecules are used toidentify similar nucleic acid molecules. Such standard conditions aredisclosed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al.,ibid., is incorporated by reference herein in its entirety (seespecifically, pages 9.31-9.62). In addition, formulae to calculate theappropriate hybridization and wash conditions to achieve hybridizationpermitting varying degrees of mismatch of nucleotides are disclosed, forexample, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkothet al., ibid., is incorporated by reference herein in its entirety.

More particularly, moderate stringency hybridization and washingconditions, as referred to herein, refer to conditions which permitisolation of nucleic acid molecules having at least about 70% nucleicacid sequence identity with the nucleic acid molecule being used toprobe in the hybridization reaction (i.e., conditions permitting about30% or less mismatch of nucleotides). High stringency hybridization andwashing conditions, as referred to herein, refer to conditions whichpermit isolation of nucleic acid molecules having at least about 80%nucleic acid sequence identity with the nucleic acid molecule being usedto probe in the hybridization reaction (i.e., conditions permittingabout 20% or less mismatch of nucleotides). Very high stringencyhybridization and washing conditions, as referred to herein, refer toconditions which permit isolation of nucleic acid molecules having atleast about 90% nucleic acid sequence identity with the nucleic acidmolecule being used to probe in the hybridization reaction (i.e.,conditions permitting about 10% or less mismatch of nucleotides). Asdiscussed above, one of skill in the art can use the formulae inMeinkoth et al., ibid. to calculate the appropriate hybridization andwash conditions to achieve these particular levels of nucleotidemismatch. Such conditions will vary, depending on whether DNA:RNA orDNA:DNA hybrids are being formed. Calculated melting temperatures forDNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particularembodiments, stringent hybridization conditions for DNA:DNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 20° C. and about 35° C. (lower stringency),more preferably, between about 28° C. and about 40° C. (more stringent),and even more preferably, between about 35° C. and about 45° C. (evenmore stringent), with appropriate wash conditions. In particularembodiments, stringent hybridization conditions for DNA:RNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 30° C. and about 45° C., more preferably,between about 38° C. and about 50° C., and even more preferably, betweenabout 45° C. and about 55° C., with similarly stringent wash conditions.These values are based on calculations of a melting temperature formolecules larger than about 100 nucleotides, 0% formamide and a G+Ccontent of about 40%. Alternatively, T_(m) can be calculated empiricallyas set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general,the wash conditions should be as stringent as possible, and should beappropriate for the chosen hybridization conditions. For example,hybridization conditions can include a combination of salt andtemperature conditions that are approximately 20-25° C. below thecalculated T_(m) of a particular hybrid, and wash conditions typicallyinclude a combination of salt and temperature conditions that areapproximately 12-20° C. below the calculated T_(m) of the particularhybrid. One example of hybridization conditions suitable for use withDNA:DNA hybrids includes a 2-24 hour hybridization in 6×SSC (50%formamide) at about 42° C., followed by washing steps that include oneor more washes at room temperature in about 2×SSC, followed byadditional washes at higher temperatures and lower ionic strength (e.g.,at least one wash as about 37° C. in about 0.1×-0.5×SSC, followed by atleast one wash at about 68° C. in about 0.1×-0.5×SSC).

In another embodiment of the invention, a homologue of a prelamin Aprotein or a pre peptide can include at least one modification to aspecific amino acid residue of the wild-type sequence, wherein theresulting homologue preferably retains a biological activity of thewild-type protein or peptide. Particularly preferred modificationsinclude at least one substitution, deletion, or insertion of an aminoacid residue for an amino acid residue that does not, or is predictednot to, substantially affect the biological activity of the protein.Referring to FIG. 2, the present inventor has aligned the prelamin A prepeptide (including the ultimately cleaved −AAX motif from prelamin A,described above) to show the conserved amino acid positions relative tothe human sequence. Based on such an alignment, one of skill in the artcan readily predict which amino acid positions are most likely totolerate substitution, modification, insertion or deletion, and whethersubstitutions or additions should be conservative or less conservative.

For example, from the alignment provided in FIG. 2, it is clear that thehuman sequence (positions 647-664 of SEQ ID NO:4; represented by SEQ IDNO:20) is most closely related to the mouse (positions 648-665 of SEQ IDNO:9; represented by SEQ ID NO:21) and chicken sequence (positions638-657 of SEQ ID NO:11; represented by SEQ ID NO:22), and less so tofrog (positions 648-665 of SEQ ID NO:13; represented by SEQ ID NO:23)and fish (positions 640-659 of SEQ ID NO:15; represented by SEQ IDNO:24), as would be expected based on taxonomy. With reference to thehuman sequence shown in FIG. 2, since the amino acid position ofrelative to human T9 (threonine at position 9 of SEQ ID NO:20) is notconserved in any species, the substitutions of serine, Arginine®) andglutamic acid (Q) for this amino acid sequence of other species areunlikely to have an effect and therefore, this position is likely totolerate a variety of substitutions or other modifications. Thesubstitutions seen in chicken, frog and fish for human S5, S6, Q10 andS11 are more intermediate in terms of the type of substitution at thisposition between species, and so one could make more conservative, butnot necessarily very conservative, substitutions or modifications atthese positions with a reasonable expectation of avoiding significantlyaltering protein activity or processing. The alignment indicates thatmodifications could also be made at positions relative to human L1 andL2. However, since the differences among species at these positions arevery conservative (e.g., a valine or isoleucine for a leucine), onewould preferably limit modifications at this position to the mostconservative possibilities (e.g., one would typically avoid substitutionof a polar or charged amino acid at these aliphatic positions, but favorsubstitutions of other aliphatic amino acids such as valine orisoleucine for the leucine residue). The positions relative to human G3,N4, P7, R8, P12, Q13, N14, C15, S16, I17 and M18 are conserved in 4 ofthe 5 species, or in all 5 species. Substitutions in these amino acidswould be the most likely to affect protein activity and/or processing,although as discussed below, substitutions or modifications at thesepositions are not excluded in the present invention.

In general, one could use the following guidelines with reference to thehuman sequence (SEQ ID NO:20). L1 and L2 are conservatively substitutedamong other species and so good choices for substitution would be otheraliphatic amino acids. G3 is only non-conservatively substituted inzebrafish, and would be an unlikely choice for substitutions that wouldnot affect activity. N4 is only nonconservatively substituted in chickenand would be a weak choice. S5 is conservatively substituted in chicken,zebrafish and frog, and the S6 is nonconservatively substituted in thesethree species. Therefore, both serines (S5 and S6) would be intermediatesites for substitutions. P7 and R8 are only non-conservativelysubstituted in Xenopus and would be weak targets, while the following T9is nonconservatively substituted in all 4 species, making it thestrongest target for substitutions that are not predicted to affectactivity. Q10 has nonconservative substitutions in zebrafish and frog,making it an intermediate candidate, while S11 is nonconservativelysubstituted in chick, fish and frog, making it an intermediate candidateas well. P12 has a conservative substitution to an S in mouse only,indicating this particular amino acid change may not affect activity,but other changes at this residue would be predicted to affect activity.Q13 has a conservative substitution in zebrafish only, making it a poorchoice, and the following N14 has a nonconservative substitution inchicken only, indicating it is not a preferred position forsubstitutions. The final CSIM (positions 15-18 of SEQ ID NO:20 in FIG.2, corresponding to positions 661-664 of SEQ ID NO:4) is the CAAX motif,and is conserved through all species, indicating it is not normallymodified. However, because the −AAX motif is known to be degenerateregarding the ability to direct farnesylation, the present inventorenvisions the possibility of making substitutions in these amino acids,particularly with regard to embodiments directed to modifying thefarnesylation processing steps of prelamin A.

Finally, it is to be understood that while positions that contain themost variability across species are the most likely to be mutatedwithout effect, any substitution which occurs between species may beconservative functionally. Therefore, even though there are only singlesubstitutions in each of the P12, Q13 and N14 amino acids at the end ofthe human sequence (SEQ ID NO:20), one might want to introduce thesesingle amino acid substitutions in the human sequence because they havelow probabilities of affecting activity.

Preferred amino acid residues of the human prelamin A pre peptidesequence for modification include, but are not limited to: 1, 2, 5, 6,9, 10, 11, 12, 13 and/or 14, with modifications at positions 1, 2, 5, 6,9, 10, 11 and/or 12 being more preferred, and modifications at positions1, 2, 5, 6, 9, 10 and/or 11 being particularly preferred.

Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine and leucine;aspartic acid, glutamic acid, asparagine, and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine.Substitutions may also be made on the basis of conserved hydrophobicityor hydrophilicity (Kyte and Doolittle, J. Mol. Biol. (1982) 157:105-132), or on the basis of the ability to assume similar polypeptidesecondary structure (Chou and Fasman, Adv. Enzymol. (1978) 47: 45-148,1978).

In another aspect of the invention, it is desirable to produce ahomologue of prelamin A that is processing deficient. In this aspect,preferred amino acid residues for modification include, but are notlimited to (with reference to SEQ ID NO:4), any residues that are rarelysubstituted across species, Arg60, Leu85, Glu203, Arg89, Asn195, Arg377,Tyr646, G649, N650, P653, R654, P658, Q659, N660, Cys661, S662, I663and/or M664. It is to be understood that modifications are not limitedto these positions of SEQ ID NO:4, as one of skill in the art willreadily be able to select other positions that are likely to tolerate atleast a conservative amino acid substitution, if not moderate to anyamino acid substitution. In one aspect, the amino acids are substitutedfor different amino acid residues as follows: Arg60Gly, Lue85Arg,Glu203Gly, Arg89Leu, Asn19Lys, and Arg377His.

The minimum size of a protein and/or homologue of the present inventionis, in one aspect, a size sufficient to have the requisite biologicalactivity, including agonist or antagonist activity, or sufficient toserve as an antigen for the generation of an antibody or as a target ordetectable reagent in an in vitro assay. In one embodiment, a prepeptide of the present invention is at least about 8 amino acids inlength, or at least about 9 amino acids in length, or at least about 10amino acids in length, or at least about 11 amino acids in length, or atleast about 12 amino acids in length, or at least about 13 amino acidsin length, or at least about 14 amino acids in length, or at least about15 amino acids in length. There is no limit, other than a practicallimit, on the maximum size of such a protein in that the protein caninclude a portion of a pre peptide or a full-length pre peptide, plusadditional sequence (e.g., a fusion protein sequence), if desired.

In one embodiment, a prelamin A protein of the present invention is atleast about 8 amino acids in length (e.g., suitable for an antibodyepitope or as a detectable reagent in an assay), or at least about 25amino acids in length, or at least about 50 amino acids in length, or atleast about 100 amino acids in length, or at least about 150 amino acidsin length, or at least about 200 amino acids in length, or at leastabout 250 amino acids in length, or at least about 300 amino acids inlength, or at least about 350 amino acids in length, or at least about400 amino acids in length, or at least about 450 amino acids in length,or at least about 500 amino acids in length, or at least about 550 aminoacids in length, or at least about 600 amino acids in length. Again,there is no limit, other than a practical limit, on the maximum size ofsuch a protein in that the protein can include a portion of a prelamin Aprotein or a full-length prelamin A protein, plus additional sequence(e.g., a fusion protein sequence), if desired.

Another embodiment of the invention relates to a fragment of prelamin Aconsisting essentially of a domain of prelamin A that has inter-nucleartransport domain biological activity, or a biologically active homologuethereof. The present inventor has shown that prelamin A is rapidlytransferred between nuclei within the myocyte, increasing its efficacy.The use of the inter-nuclear transport domain as a targeting moiety forother pharmaceuticals would increase their efficacy without introducingtoxicity. To identify the exact sequence of the transport domain will bestraightforward. Briefly, in order to identify the lamin A proteinsequences responsible for internuclear transport, deletion-mappingexperiments will be performed on the wild type prelamin A GFP-fusionprotein construct. Initially, restriction enzymes will be used to createlarge, overlapping deletions in the prelamin A cDNA sequence. Forexample, C2C12 myoblasts will be transfected with the deletionconstructs and induced to differentiate. Protein regions responsible forinternuclear transport will be identified as those which preventinternuclear transport of the GFP fusion protein when deleted. Once theregion encoding the transport domain is identified, site-directedmutagenesis will be used to delineate the minimal protein sequencenecessary for internuclear protein transport.

Complementary experiments will be performed in which the regions deletedfrom the prelamin A cDNA in the experiments described above will becloned downstream of GFP coding sequences in a plasmid which does notcontain any other prelamin A coding sequences. The ability of the clonedprelamin A subsequences to direct internuclear transport when fuseddirectly to GFP will be assessed by transfecting C2C12 myoblasts withthe plasmid expression constructs and examining the expression of GFPwithin the nuclei of myotubes. These experiments will confirm thefunctional role of protein transport sequences identified by deletionmapping, and allow for the analysis of peptide sequences that may resultin protein degradation when deleted from the full-length prelamin Aprotein.

The present invention also includes a fusion protein that includes a prepeptide-, prelamin A-, or prelamin A inter-nuclear transportdomain-containing segment (i.e., an amino acid sequence for a prepeptide, a prelamin A protein, or a prelamin A inter-nuclear transportdomain according to the present invention, including homologues andfragments thereof) attached to one or more fusion segments. Suitablefusion segments for use with the present invention include, but are notlimited to, segments that can: enhance a protein's stability; provideother desirable biological activity (e.g., a therapeutic protein/peptideto be delivered to a site); and/or assist with the purification of theprotein (e.g., by affinity chromatography). A suitable fusion segmentcan be a domain of any size that has the desired function (e.g., impartsincreased stability, solubility, biological activity; and/or simplifiespurification of a protein). Fusion segments can be joined to aminoand/or carboxyl termini of the pre peptide-, prelamin A-, or prelamin Ainter-nuclear transport domain-containing segment of the protein and canbe susceptible to cleavage in order to enable straight-forward recoveryof the desired protein. Fusion proteins are preferably produced byculturing a recombinant cell transfected with a fusion nucleic acidmolecule that encodes a protein including the fusion segment attached toeither the carboxyl and/or amino terminal end of a pre peptide-,prelamin A-, or prelamin A inter-nuclear transport domain-containingsegment.

In one aspect, a prelamin A inter-nuclear transport domain is atherapeutic composition for promoting myoblast activation and growth orregeneration of cardiac or skeletal muscle, comprising an isolatedpeptide consisting essentially of an isolated fragment of SEQ ID NO:4with inter-nuclear transport domain activity or a biologically activehomologue thereof. The peptide is fused to a therapeutic protein forpromoting myoblast activation and growth or regeneration of cardiac orskeletal muscle, or to a drug for use in the treatment of heart andskeletal muscle diseases. Fusion to the transport domain will likelyincrease the efficacy of drugs with nuclear functions, and may alsoincrease the distribution of drugs that do not have specifically nuclearfunctions. There are currently no treatments for muscular dystrophies,although gene therapy has been proposed using emerin, dystrophin andother genes known to be mutated in these diseases. Therefore, fusion ofthe prelamin A transport domain to such genes could enhance theseputative therapies. For cardiomyopathies, one treatment currentlyincludes the use of “beta blockers” to block the beta adrenergicresponse pathway. However, the exact mechanism of action is not known,as there is some evidence that certain beta-blockers (carvedilol) may beefficacious due to their antioxidant activity. As beta-blockers are theprimary therapy for cardiomyopathies, the present invention includes thefusion of the prelamin A transport domain to such drugs. The inventionintends to encompass the use of any drugs that are used to treat otherheart diseases, which could benefit from the transport domain.

In one embodiment of the present invention, any of the amino acidsequences described herein can be produced with from at least one, andup to about 20, additional heterologous amino acids flanking each of theC- and/or N-terminal ends of the specified amino acid sequence (thespecified amino acid sequence being, for example, SEQ ID NO:2, SEQ IDNO:4, a biologically active fragment thereof or a biologically activehomologue thereof). The resulting protein or polypeptide can be referredto as “consisting essentially of” the specified amino acid sequence.According to the present invention, the heterologous amino acids are asequence of amino acids that are not naturally found (i.e., not found innature, in vivo) flanking the specified amino acid sequence, or that arenot related to the function of the specified amino acid sequence, orthat would not be encoded by the nucleotides that flank the naturallyoccurring nucleic acid sequence encoding the specified amino acidsequence as it occurs in the gene, if such nucleotides in the naturallyoccurring sequence were translated using standard codon usage for theorganism from which the given amino acid sequence is derived. Similarly,the phrase “consisting essentially of”, when used with reference to anucleic acid sequence herein, refers to a nucleic acid sequence encodinga specified amino acid sequence that can be flanked by from at leastone, and up to as many as about 60, additional heterologous nucleotidesat each of the 5′ and/or the 3′ end of the nucleic acid sequenceencoding the specified amino acid sequence. The heterologous nucleotidesare not naturally found (i.e., not found in nature, in vivo) flankingthe nucleic acid sequence encoding the specified amino acid sequence asit occurs in the natural gene or do not encode a protein that imparts anadditional function to the protein or changes the function of theprotein having the specified amino acid sequence.

Another embodiment of the present invention relates to a compositioncomprising at least about 500 ng, and preferably at least about 1 μg,and more preferably at least about 5 μg, and more preferably at leastabout 10 μg, and more preferably at least about 25 μg, and morepreferably at least about 50 μg, and more preferably at least about 75μg, and more preferably at least about 100 μg, and more preferably atleast about 250 μg, and more preferably at least about 500 μg, and morepreferably at least about 750 μg, and more preferably at least about 1mg, and more preferably at least about 5 mg, of an isolated pre peptideor a prelamin A protein comprising any of the proteins, fragmentsthereof or homologues thereof discussed herein (including, for example,a fragment having the prelamin A inter-nuclear transport domainbiological activity). Such a composition of the present invention caninclude any carrier with which the protein is associated by virtue ofthe protein preparation method, a protein purification method, or apreparation of the protein for use in an in vitro, ex vivo, or in vivomethod according to the present invention. For example, such a carriercan include any suitable excipient, buffer and/or delivery vehicle, suchas a pharmaceutically acceptable carrier (discussed below), which issuitable for combining with the protein so that the protein can be usedin vitro, ex vivo or in vivo according to the present invention.Compositions of the invention, including therapeutic compositions, arediscussed in detail below.

Another embodiment of the invention relates to a processing deficientprelamin A peptide, wherein the processing deficient prelamin A peptideconsists essentially of an amino acid sequence that differs from SEQ IDNO:4 (or a functional allelic variant thereof) by at least onesubstitution, deletion or insertion that results in a decrease in aprelamin A or prelamin A pre peptide biological activity. Such activitycan include, but is not limited to: (a) prelamin A processing to releasea prelamin A pre peptide (e.g., SEQ ID NO:2 or a biologically activehomologue thereof); (b) prelamin A pre peptide signal transduction; (c)synchronization of intercellular signaling with changes in lamin Alocalization and nuclear lamina morphology that occur early in myoblastdifferentiation; (d) synchronization of transcriptional regulation ofmuscle-specific genes or cell cycle arrest that occurs concomitant withmyoblast differentiation; (e) formation of normal nuclear laminastructure; and (f) induction of myoblast activation and differentiation.In one embodiment, the processing deficient prelamin A peptide consistsessentially of an amino acid sequence that differs from SEQ ID NO:4 by asubstitution of an amino acid residue in SEQ ID NO:4 selected from thegroup of: any amino acid that is rarely (e.g., less than 20% of thetime) substituted across species, or Arg60, Leu85, Glu203, Arg89,Asn195, Arg377, Tyr646, G649, N650, P653, R654, P658, Q659, N660,Cys661, S662, I663 and/or M664. In another embodiment, the substitutionis selected from the group of: Arg60Gly, Lue85Arg, Glu203Gly, Arg89Leu,Asn19Lys, and Arg377His. Also encompassed by the invention are isolatedcells transfected with any of the processing deficient prelamin Aproteins described herein.

Further embodiments of the present invention include nucleic acidmolecules that encode any of the above-identified proteins, including ahomologue or fragment thereof. In one embodiment, a nucleic acidmolecule encoding pre peptide includes the nucleic acid sequencerepresented by SEQ ID NO:1, fragments thereof, or nucleic acid moleculesencoding homologues of SEQ ID NO:2 as described herein. Nucleic acidmolecules encoding prelamin A include the nucleic acid sequencerepresented by SEQ ID NO:3, fragments thereof, or nucleic acid moleculesencoding homologues of SEQ ID NO:4 as described herein. In accordancewith the present invention, an isolated polynucleotide, or an isolatednucleic acid molecule, is a nucleic acid molecule that has been removedfrom its natural milieu (i.e., that has been subject to humanmanipulation), its natural milieu being the genome or chromosome inwhich the nucleic acid molecule is found in nature. As such, “isolated”does not necessarily reflect the extent to which the nucleic acidmolecule has been purified, but indicates that the molecule does notinclude an entire genome or an entire chromosome in which the nucleicacid molecule is found in nature. An isolated nucleic acid molecule caninclude a gene or a portion of a gene (e.g., the regulatory region orpromoter). An isolated nucleic acid molecule that includes a gene is nota fragment of a chromosome that includes such gene, but rather includesthe coding region and regulatory regions associated with the gene, butno additional genes naturally found on the same chromosome. An isolatednucleic acid molecule can also include a specified nucleic acid sequenceflanked by (i.e., at the 5′ and/or the 3′ end of the sequence)additional nucleic acids that do not normally flank the specifiednucleic acid sequence in nature (i.e., heterologous sequences). Isolatednucleic acid molecule can include DNA, RNA (e.g., mRNA), or derivativesof either DNA or RNA (e.g., cDNA). Although the phrase “nucleic acidmolecule” primarily refers to the physical nucleic acid molecule and thephrase “nucleic acid sequence” primarily refers to the sequence ofnucleotides on the nucleic acid molecule, the two phrases can be usedinterchangeably, especially with respect to a nucleic acid molecule, ora nucleic acid sequence, being capable of encoding a protein.Preferably, an isolated nucleic acid molecule of the present inventionis produced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis.

Isolated nucleic acid molecules include natural nucleic acid moleculesand homologues thereof, including, but not limited to modified (mutated)nucleic acid molecules in which, as compared to the natural or wild-typesequence, nucleotides have been inserted, deleted, substituted, and/orinverted in such a manner that such modifications (mutations) result ina nucleic acid sequence that encodes a desired homologue of a protein asdescribed herein. A nucleic acid molecule homologue (e.g., a nucleicacid molecule encoding a protein homologue of the present invention) canbe produced using a number of methods known to those skilled in the art(see, for example, Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Labs Press, 1989). For example, wild-typenucleic acid molecules can be modified or nucleic acid moleculesencoding modified proteins can be created using a variety of techniquesincluding, but not limited to, classic mutagenesis techniques andrecombinant DNA techniques, such as site-directed mutagenesis, chemicaltreatment of a nucleic acid molecule to induce mutations, restrictionenzyme cleavage of a nucleic acid fragment, ligation of nucleic acidfragments, PCR amplification and/or mutagenesis of selected regions of anucleic acid sequence, synthesis of oligonucleotide mixtures andligation of mixture groups to “build” a mixture of nucleic acidmolecules and combinations thereof. Nucleic acid molecule homologues canbe selected from a mixture of modified nucleic acids, for example, byscreening for the function of the protein encoded by the nucleic acidand/or by hybridization with a wild-type gene.

The minimum size of a nucleic acid molecule of the present invention isa size sufficient to form a probe or oligonucleotide primer that iscapable of forming a stable hybrid (e.g., under moderate, high or veryhigh stringency conditions, discussed in detail above) with thecomplementary sequence of a nucleic acid molecule useful in the presentinvention, or of a size sufficient to encode an amino acid sequence fora protein of the invention. The nucleic acid molecule may also includeregulatory regions, linker sequences, vector sequence or other sequenceas necessary to provide a nucleic acid molecule according to the presentinvention. The minimal size of a nucleic acid molecule that is used asan oligonucleotide primer or as a probe is typically at least about 12to about 15 nucleotides in length if the nucleic acid molecules areGC-rich and at least about 15 to about 18 bases in length if they areAT-rich. An oligonucleotide preferably ranges from about 5 to about 50or about 500 nucleotides, including any size between about 5 and about500 in whole integers (i.e., 5, 6, 7, 8, 9, . . . 34, 35, 36, . . . 200,201, 202, . . . 500), and more preferably from about 10 to about 40nucleotides, and most preferably from about 15 to about 40 nucleotidesin length. There is no limit, other than a practical limit, on themaximal size of a nucleic acid molecule of the present invention, inthat the nucleic acid molecule can include a sequence sufficient toencode the proteins of the invention and since the size of the nucleicacid molecule encoding such proteins can be dependent on nucleic acidcomposition and whether regulatory regions and/or other sequence areincluded (e.g., linkers, vector sequence, etc.).

Particularly preferred nucleic acid molecules according to the presentinvention include nucleic acid molecules comprising, consistingessentially of, or consisting of, nucleic acid sequences encoding any ofthe above-described amino acid sequences, including homologues thereof.In one embodiment, such a nucleic acid sequence includes an a nucleicacid sequence that is at least about 55% identical, or at least about60% identical, or at least about 65% identical, or at least about 70%identical, or at least about 75% identical, or at least about 80%identical, or at least about 85% identical, or at least about 90%identical, or at least about 95% identical, or at least about 96%identical, or at least about 97% identical, or at least about 98%identical, or at least about 99% identical to SEQ ID NO:1 or SEQ IDNO:3, or to any of the other nucleic acid sequences described herein,over the full length of any of such sequences. Particularly preferrednucleic acid sequences include, but are not limited to, SEQ ID NO:1, SEQID NO:3, or fragments of such sequences, including a nucleic acidsequence encoding an isolated fragment of SEQ ID NO:4 with inter-nucleartransport domain biological activity or a biologically active homologuethereof. Additionally, fragments and other homologues of such sequencescorresponding to the encoded amino acid sequences described above arealso included. In one embodiment, the nucleic acid molecule does notencode a protein with biological activity, but is an oligonucleotideprobe or primer (described previously herein).

One embodiment of the present invention relates to a recombinant nucleicacid molecule which comprises any of the isolated nucleic acid moleculesdescribed above which is operatively linked to at least onetranscription control sequence. More particularly, according to thepresent invention, a recombinant nucleic acid molecule typicallycomprises a recombinant vector and an isolated nucleic acid molecule asdescribed herein. According to the present invention, a recombinantvector is an engineered (i.e., artificially produced) nucleic acidmolecule that is used as a tool for manipulating a nucleic acid sequenceof choice and/or for introducing such a nucleic acid sequence into ahost cell. The recombinant vector is therefore suitable for use incloning, sequencing, and/or otherwise manipulating the nucleic acidsequence of choice, such as by expressing and/or delivering the nucleicacid sequence of choice into a host cell to form a recombinant cell.Such a vector typically contains heterologous nucleic acid sequences,that is, nucleic acid sequences that are not naturally found adjacent tonucleic acid sequence to be cloned or delivered, although the vector canalso contain regulatory nucleic acid sequences (e.g., promoters,untranslated regions) which are naturally found adjacent to nucleic acidsequences of the present invention or which are useful for expression ofthe nucleic acid molecules of the present invention (discussed in detailbelow). The vector can be either RNA or DNA, either prokaryotic oreukaryotic, and typically is a plasmid. The vector can be maintained asan extrachromosomal element (e.g., a plasmid) or it can be integratedinto the chromosome of a recombinant host cell. The entire vector canremain in place within a host cell, or under certain conditions, theplasmid DNA can be deleted, leaving behind the nucleic acid molecule ofthe present invention. An integrated nucleic acid molecule can be underchromosomal promoter control, under native or plasmid promoter control,or under a combination of several promoter controls. Single or multiplecopies of the nucleic acid molecule can be integrated into thechromosome. A recombinant vector of the present invention can contain atleast one selectable marker.

In one embodiment, a recombinant vector used in a recombinant nucleicacid molecule of the present invention is an expression vector. As usedherein, the phrase “expression vector” is used to refer to a vector thatis suitable for production of an encoded product (e.g., a protein ofinterest). In this embodiment, a nucleic acid sequence encoding theproduct to be produced (e.g., a prelamin A pre peptide) is inserted intothe recombinant vector to produce a recombinant nucleic acid molecule.The nucleic acid sequence encoding the protein to be produced isinserted into the vector in a manner that operatively links the nucleicacid sequence to regulatory sequences in the vector which enable thetranscription and translation of the nucleic acid sequence within therecombinant host cell.

Typically, a recombinant nucleic acid molecule includes at least onenucleic acid molecule of the present invention operatively linked to oneor more expression control sequences. As used herein, the phrase“recombinant molecule” or “recombinant nucleic acid molecule” primarilyrefers to a nucleic acid molecule or nucleic acid sequence operativelylinked to an expression control sequence, but can be usedinterchangeably with the phrase “nucleic acid molecule”, when suchnucleic acid molecule is a recombinant molecule as discussed herein.According to the present invention, the phrase “operatively linked”refers to linking a nucleic acid molecule to an expression controlsequence (e.g., a transcription control sequence and/or a translationcontrol sequence) in a manner such that the molecule is able to beexpressed when transfected (i.e., transformed, transduced, transfected,conjugated or conduced) into a host cell. Transcription controlsequences are sequences which control the initiation, elongation, ortermination of transcription. Particularly important transcriptioncontrol sequences are those which control transcription initiation, suchas promoter, enhancer, operator and repressor sequences. Suitabletranscription control sequences include any transcription controlsequence that can function in a host cell or organism into which therecombinant nucleic acid molecule is to be introduced.

Preferred promoters to use in a recombinant nucleic acid moleculeaccording to the invention include any promoter which can function inthe cardiac or skeletal muscle tissue. Such promoters include, but arenot limited to, a cardiac-specific promoter, a muscle-specific promoter,and a prelamin A promoter. In one aspect, the promoter is a myosin heavychain promoter.

Recombinant nucleic acid molecules of the present invention can alsocontain additional expression control and other regulatory sequences,such as translation regulatory sequences, origins of replication, andother regulatory sequences that are compatible with the recombinantcell. In one embodiment, a recombinant molecule of the presentinvention, including those which are integrated into the host cellchromosome, also contains secretory signals (i.e., signal segmentnucleic acid sequences) to enable an expressed protein to be secretedfrom the cell that produces the protein. Suitable signal segmentsinclude a signal segment that is naturally associated with the proteinto be expressed or any heterologous signal segment capable of directingthe secretion of the protein according to the present invention. Inanother embodiment, a recombinant molecule of the present inventioncomprises a leader sequence to enable an expressed protein to bedelivered to and inserted into a membrane of a host cell. Suitableleader sequences include a leader sequence that is naturally associatedwith the protein, or any heterologous leader sequence capable ofdirecting the delivery and insertion of the protein to a membrane of acell.

According to the present invention, the term “transfection” is used torefer to any method by which an exogenous nucleic acid molecule (i.e., arecombinant nucleic acid molecule) can be inserted into a cell. The term“transformation” can be used interchangeably with the term“transfection” when such term is used to refer to the introduction ofnucleic acid molecules into microbial cells. In microbial systems, theterm “transformation” is used to describe an inherited change due to theacquisition of exogenous nucleic acids by the microorganism and isessentially synonymous with the term “transfection.” However, in animalcells, transformation has acquired a second meaning which can refer tochanges in the growth properties of cells in culture (described above)after they become cancerous, for example. Therefore, to avoid confusion,the term “transfection” is preferably used with regard to theintroduction of exogenous nucleic acids into animal cells, and is usedherein to generally encompass transfection of animal cells andtransformation of microbial cells, to the extent that the terms pertainto the introduction of exogenous nucleic acids into a cell. Therefore,transfection techniques include, but are not limited to, transformation,particle bombardment, diffusion, active transport, bath sonication,electroporation, microinjection, lipofection, adsorption, infection andprotoplast fusion.

One or more recombinant molecules of the present invention can be usedto produce an encoded product (e.g., a prelamin A protein or a prelaminA pre peptide) of the present invention. In one embodiment, an encodedproduct is produced by expressing a nucleic acid molecule as describedherein under conditions effective to produce the protein. A preferredmethod to produce an encoded protein is by transfecting a host cell withone or more recombinant molecules to form a recombinant cell. Suitablehost cells to transfect include, but are not limited to, any bacterial,fungal (e.g., yeast), insect, plant or animal cell that can betransfected. Host cells can be either untransfected cells or cells thatare already transfected with at least one other recombinant nucleic acidmolecule.

In one embodiment, one or more protein(s) expressed by an isolatednucleic acid molecule of the present invention are produced by culturinga cell that expresses the protein (i.e., a recombinant cell orrecombinant host cell) under conditions effective to produce theprotein. In some instances, the protein may be recovered, and in others,the cell may be harvested in whole (e.g., for ex vivo administration),either of which can be used in a composition. In some instances, theprotein may be expressed in a host cell in vivo (e.g., via genetherapy). A preferred cell to culture is any suitable host cell asdescribed above. Effective in vitro or ex vivo culture conditionsinclude, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein productionand/or recombination. An effective medium refers to any medium in whicha given host cell is typically cultured. Such medium typically comprisesan aqueous medium having assimilable carbon, nitrogen and phosphatesources, and appropriate salts, minerals, metals and other nutrients,such as vitamins. Cells can be cultured in conventional fermentationbioreactors, shake flasks, test tubes, microtiter dishes, and petriplates. Culturing can be carried out at a temperature, pH and oxygencontent appropriate for a recombinant cell. Such culturing conditionsare within the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell; be secreted into the culture medium; be secreted intoa space between two cellular membranes; or be retained on the outersurface of a cell membrane. The phrase “recovering the protein” refersto collecting the whole culture medium containing the protein and neednot imply additional steps of separation or purification. Proteinsproduced according to the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.

Proteins produced according to the present invention are preferablyretrieved in “substantially pure” form. As used herein, “substantiallypure” refers to a purity that allows for the effective use of theprotein in vitro, ex vivo or in vivo according to the present invention.For a protein to be useful in an in vitro, ex vivo or in vivo methodaccording to the present invention, it is typically substantially freeof contaminants, other proteins and/or chemicals that might interfere orthat would interfere with its use in a method disclosed by the presentinvention, or that at least would be undesirable for inclusion with theprotein (including homologues) when it is used in a method disclosed bythe present invention. assays, preparation of therapeutic compositions,administration in a therapeutic composition, and all other methodsdisclosed herein. Preferably, a “substantially pure” protein, asreferenced herein, is a protein that can be produced by any method(i.e., by direct purification from a natural source, recombinantly, orsynthetically), and that has been purified from other protein componentssuch that the protein comprises at least about 80% weight/weight of thetotal protein in a given composition (e.g., a prelamin A protein isabout 80% of the total protein in a solution/composition/buffer), andmore preferably, at least about 85%, and more preferably at least about90%, and more preferably at least about 91%, and more preferably atleast about 92%, and more preferably at least about 93%, and morepreferably at least about 94%, and more preferably at least about 95%,and more preferably at least about 96%, and more preferably at leastabout 97%, and more preferably at least about 98%, and more preferablyat least about 99%, weight/weight of the total protein in a givencomposition.

It will be appreciated by one skilled in the art that use of recombinantDNA technologies can improve control of expression of transfectednucleic acid molecules by manipulating, for example, the number ofcopies of the nucleic acid molecules within the host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Additionally, thepromoter sequence might be genetically engineered to improve the levelof expression as compared to the native promoter. Recombinant techniquesuseful for controlling the expression of nucleic acid molecules include,but are not limited to, integration of the nucleic acid molecules intoone or more host cell chromosomes, addition of vector stabilitysequences to plasmids, substitutions or modifications of transcriptioncontrol signals (e.g., promoters, operators, enhancers), substitutionsor modifications of translational control signals (e.g., ribosomebinding sites, Shine-Dalgamo sequences), modification of nucleic acidmolecules to correspond to the codon usage of the host cell, anddeletion of sequences that destabilize transcripts.

In one embodiment of the invention, the recombinant nucleic acidmolecule comprises a viral vector. A viral vector includes an isolatednucleic acid molecule of the present invention integrated into a viralgenome or portion thereof, in which the nucleic acid molecule ispackaged in a viral coat that allows entrance of DNA into a cell. Anumber of viral vectors can be used, including, but not limited to,those based on alphaviruses, poxviruses, adenoviruses, herpesviruses,lentiviruses, adeno-associated viruses and retroviruses.

The isolated nucleic acid molecules of the present invention, as well asthe proteins produced by such molecules are all useful in variouscompositions of the invention. For example, in one embodiment, theisolated nucleic acid molecule (preferably as part of a recombinantnucleic acid molecule) is useful as for gene therapy, whereinadministration of the nucleic acid molecule to an animal results intransfection of host cells of the animal with the molecule andexpression of the protein(s) expressed by the molecule. As discussedabove, nucleic acids encoding the prelamin A pre peptide or prelamin Aare excellent candidates for gene therapy of cardiac and skeletal muscledisorders and degeneration. The present inventor's data shows thatprelamin A is rapidly transferred between the multiple nuclei within amyocyte, and affects the morphology and organization of the transfectedmyocytes as well as that of adjacent untransfected myocytes. In anotherembodiment, the isolated nucleic acid molecule is used to produce theencoded protein(s) in vitro, which can then be used in a therapeuticcomposition. In yet another embodiment, the isolated nucleic acidmolecule can be used to transfect cells ex vivo and then the cells arereturned to the patient from which they were removed.

In one embodiment of the present invention, a therapeutic composition(comprising a nucleic acid or a protein) comprises a pharmaceuticallyacceptable carrier, which includes pharmaceutically acceptableexcipients and/or delivery vehicles, for delivering the recombinantnucleic acid molecule or the proteins to a patient. As used herein, apharmaceutically acceptable carrier refers to any substance or vehiclesuitable for delivering a therapeutic composition useful in atherapeutic method of the present invention (described below) to asuitable in vivo or ex vivo site. When a nucleic acid molecule is in thecomposition, preferred pharmaceutically acceptable carriers are capableof maintaining the nucleic acid molecule in a form that, upon arrival ofthe nucleic acid molecule to a target cell or tissue, the nucleic acidmolecule is capable of entering the cell and being expressed by thecell, whereby the expressed protein can perform one or more biologicalactivities of the protein as described previously herein. When thecomposition comprises a protein, preferred pharmaceutically acceptablecarriers are capable of maintaining the protein composition in a formthat, upon arrival of the protein composition to a target cell ortissue, the proteins are capable of performing one or more biologicalfunctions of the protein as discussed above at the cell or tissue site.

A pharmaceutically acceptable carrier can include a pharmaceuticallyacceptable excipient. Suitable excipients of the present inventioninclude excipients or formularies useful in a therapeutic composition.Examples of pharmaceutically acceptable excipients include, but are notlimited to water, phosphate buffered saline, Ringer's solution, dextrosesolution, serum-containing solutions, Hank's solution, other aqueousphysiologically balanced solutions, oils, esters and glycols. Aqueouscarriers can contain suitable auxiliary substances required toapproximate the physiological conditions of the recipient, for example,by enhancing chemical stability and isotonicity.

Suitable pharmaceutically acceptable carriers for nucleic acids include,but are not limited to liposomes or other lipid-containing vehicles,viral vectors, ribozymes, gold particles, poly-L-lysine/DNA-molecularconjugates, and artificial chromosomes. Natural lipid-containingdelivery vehicles include cells and cellular membranes. Artificiallipid-containing delivery vehicles include liposomes and micelles. Adelivery vehicle can be modified to target to a particular site in apatient, thereby targeting and making use of a nucleic acid molecule atthat site. Suitable modifications include manipulating the chemicalformula of the lipid portion of the delivery vehicle and/or introducinginto the vehicle a targeting agent (e.g., an antibody or peptide)capable of specifically targeting a delivery vehicle to a preferredsite, for example, a preferred cell type. It is noted, however, thatprelamin A and the prelamin A pre peptide are specific for cardiac andskeletal muscle tissue and therefore inherently will “target” theappropriate tissue and cell types. Therefore, the present invention isparticularly advantageous in that while targeting moieties can be used,they are likely not necessary to administer these proteins or peptides(or nucleic acids encoding them) in vivo.

A liposome delivery vehicle comprises a lipid composition that iscapable of delivering a nucleic acid molecule of the present invention,including naked DNA, plasmids and viral vectors, to a suitable celland/or tissue in a patient. A liposome delivery vehicle comprises alipid composition that is capable of fusing with the plasma membrane ofthe target cell to deliver the recombinant nucleic acid molecule into acell. As discussed above, liposome delivery vehicles can be modified totarget a particular site in a patient (i.e., a targeting liposome),thereby targeting and making use of a nucleic acid molecule of thepresent invention at that site. Suitable modifications includemanipulating the chemical formula of the lipid portion of the deliveryvehicle. Manipulating the chemical formula of the lipid portion of thedelivery vehicle can elicit the extracellular or intracellular targetingof the delivery vehicle. For example, a chemical can be added to thelipid formula of a liposome that alters the charge of the lipid bilayerof the liposome so that the liposome fuses with particular cells havingparticular charge characteristics. Other targeting mechanisms includetargeting a site by addition of exogenous targeting molecules (i.e.,targeting agents) to a liposome (e.g., antibodies, soluble receptors orligands). Targeting liposomes are described, for example, in Ho et al.,1986, Biochemistry 25: 5500-6; Ho et al., 1987a, J Biol Chem 262:13979-84; Ho et al., 1987b, J Biol Chem 262: 13973-8; and U.S. Pat. No.4,957,735 to Huang et al., each of which is incorporated herein byreference in its entirety).

Suitable pharmaceutically acceptable carriers for protein compositionsinclude, but are not limited to, liquid injectables or solids which canbe taken up in a suitable liquid as a suspension or solution forinjection, liquids that can be aerosolized, capsules, tablets, orliposomes. In a non-liquid formulation, the excipient can comprise, forexample, dextrose, human serum albumin, and/or preservatives to whichsterile water or saline can be added prior to administration.

One type of pharmaceutically acceptable carrier includes a controlledrelease formulation that is capable of slowly releasing a composition ofthe present invention into an animal. As used herein, a controlledrelease formulation comprises recombinant nucleic acid molecule orprotein composition of the present invention in a controlled releasevehicle. Suitable controlled release vehicles include, but are notlimited to, biocompatible polymers, other polymeric matrices, capsules,microcapsules, microparticles, bolus preparations, osmotic pumps,diffusion devices, liposomes, lipospheres, and transdermal deliverysystems.

Proteins, nucleic acids and compositions of the invention are useful ina variety of methods, including assays for the identification ofcompounds (including genes and proteins), as well as a variety oftherapeutic methods. In one embodiment, the present invention includesmethods which use nucleic acid sequences encoding prelamin A protein,prelamin A pre peptide, homologues and fragments thereof, and/orisolated cells that express such proteins, peptides and homologues(including recombinant cells and naturally occurring cells) astherapeutic reagents, screening tools and/or diagnostic tools.

Accordingly, embodiments of the present invention relate to: (1) amethod to promote myoblast activation and regeneration of damaged,degenerated or atrophied cardiac and skeletal myocytes; (2) a method tostimulate cardiac or skeletal muscle growth in a mammal; and (3) amethod to treat cardiac and skeletal muscle disorders. Each of thesemethods includes the step of administering to a patient that has acardiac or skeletal muscle disorder, an agent selected from: (a) aprelamin A protein, prelamin A pre peptide, prelamin A internucleartransport fragment (e.g., fused to a therapeutic agent), or a fragmentor homologue thereof as described previously herein; (b) a nucleic acidmolecule encoding any of such proteins, peptides, fragments, orhomologues as in (a); (c) a composition comprising any of such proteins,peptides, fragments, or nucleic acids of (a) or (b). The first method isuseful for generally promoting myoblast activation and/or regenerationof damaged, degenerated or atrophied cardiac or skeletal myocytes,whether or not the patient is suffering from a disorder that involvesthese cells. The second method is useful to promote the growth ofcardiac or skeletal muscle tissue, for example in the absence of anydamage, disorder or atrophy, as may be desirable in athletes orastronauts. The third method is useful for treating cardiac and skeletalmuscle disorders, including, but not limited to: dilated cardiomyopathy,Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy,partial lipodystrophy, axonal neuropathy, and mandibuloacral dysplasia.Another embodiment of the invention (discussed in detail below),comprises inhibiting prelamin A processing and particularly, prelamin Afarnesylation, for the treatment of muscle cell cancers or metastaticcancers thereof.

Unique features of the prelamin A “pre” peptide described in detailherein are that it is a naturally occurring, small, biologically activesignaling peptide. It would be easy to synthesize in a host cell, andparticularly, in yeast, since yeast contain all of the necessaryprocessing enzymes. In addition, the peptide would inherently becardiac- and skeletal muscle-specific in its effects, and it would haveno toxicity. The peptide would affect myocyte fusion in adults, as allhuman and mouse phenotypes are adult onset. Furthermore, the peptideexerts its effect on C2C12 and H9C2 cells, which share features with thesatellite cells involved in adult skeletal muscle repair. By analogy toyeast a-type mating pheromone, the pre sequence of pre-lamin A is anearly signaling molecule in myocyte differentiation, indicating that itwould be a highly potent and efficacious treatment. Moreover, the use ofprelamin A as a gene therapy shares these advantages and additionally,the present inventor has shown that this protein is rapidly transferredbetween nuclei within the myocyte, increasing its efficacy. Finally, theuse of the inter-nuclear transport domain as a targeting moiety forother pharmaceuticals would increase their efficacy without introducingtoxicity.

According to the present invention, the phrase “protected from adisease” refers to reducing the symptoms of the disease; reducing theoccurrence of the disease, and/or reducing the severity of the disease.Protecting a patient can refer to the ability of a therapeuticcomposition of the present invention, when administered to a patient, toprevent a disease from occurring and/or to cure or to alleviate diseasesymptoms, signs or causes. As such, to protect a patient from a diseaseincludes both preventing disease occurrence (prophylactic treatment) andtreating a patient that has a disease or that is experiencing initialsymptoms or later stage symptoms of a disease (therapeutic treatment).In particular, protecting a patient from a cardiac or skeletal muscledisease is accomplished according to the present invention byincreasing: prelamin A processing, myoblast activation (includingmyoblast cell fusion and differentiation), prelamin A pre peptide signaltransduction, and/or proper lamina formation. Protecting a patient froma muscle cell cancer or metastatic cancer thereof is accomplished by:reducing or preventing the prelamin A processing or at least thefarnesylation of muscle cell tumors, and/or causing muscle cell tumordegeneration or cell death. The term, “disease” refers to any deviationfrom the normal health of a patient and includes a state when diseasesymptoms are present, as well as conditions in which a deviation (e.g.,infection, gene mutation, genetic defect, etc.) has occurred, butsymptoms are not yet manifested.

According to the present invention, an effective administration protocol(i.e., administering a therapeutic composition in an effective manner)comprises suitable dose parameters and modes of administration thatresult in an increase in any one or more of the biological activitiesassociated with prelamin A or the prelamin A pre peptide as describedabove. Preferably, the patient is protected from the disease (e.g., bydisease prevention or by alleviating one or more symptoms of ongoingdisease). Effective dose parameters can be determined using methodsstandard in the art for a particular disease or condition. As mentionedabove, in some circumstances, the patient may not have disease, butrather muscle atrophy, some muscle cell damage, or perhaps no disease orcondition at all (e.g., in the case of an athlete). Effective doseparameters can be determined by those of skill in the art depending onthe desired effect (e.g., stimulation of growth of healthy cardiac orskeletal muscle tissue, repair or regeneration of damaged tissue, etc.).Such parameters include, for example, determination of survival rates,side effects (i.e., toxicity), progression or regression of disease, orprogress in tissue growth. In particular, the effectiveness of doseparameters of a therapeutic composition of the present invention whentreating cancer can be determined by assessing response rates. Suchresponse rates refer to the percentage of treated patients in apopulation of patients that respond with either partial or completeremission. Remission can be determined by, for example, measuring tumorsize or microscopic examination for the presence of cancer cells in atissue sample.

In accordance with the present invention, a suitable single dose size isa dose that results in regulation of the prelamin A processing pathwayand associated biological activities and effects in a patient whenadministered one or more times over a suitable time period. Doses canvary depending upon the disease being treated. For example, in thetreatment of cancer, a suitable single dose can be dependent uponwhether the cancer being treated is a primary tumor or a metastatic formof cancer. One of skill in the art can readily determine appropriatesingle dose sizes for a given patient based on the size of a patient andthe route of administration. One of skill in the art can monitor theeffectiveness of a treatment to repair damaged cardiac or skeletalmuscle tissue by measuring, for example, cell morphology, physiologicalindicators of healthy cardiac and skeletal muscle tissue, physiologicalindicators of damaged cardiac and skeletal muscle tissue (e.g., creatinekinase), and include tests such as EKG, echocardiography,catheterization, heart biopsy, MRI, motion and strength tests, andmuscle biopsies.

In one aspect of the invention, a suitable single dose of a therapeuticcomposition of the present invention is an amount that, whenadministered by any route of administration, increases at least oneaspect of the prelamin A processing pathway or downstream effects(described previously), as compared to a patient which has not beenadministered with the therapeutic composition of the present invention(i.e., a control patient), as compared to the patient prior toadministration of the composition, or as compared to a standardestablished for the particular disease, patient type and composition. Inthe case of cancer, a suitable single dose is an amount that decreasesat least one symptom of the cancer, as compared to the same controls.Preferably, a suitable single dose of a therapeutic composition againsta tumor is an amount that is sufficient to reduce, stop the growth of,and preferably eliminate, the tumor following administration of thecomposition into the tissue of the patient that has cancer.

It will be obvious to one of skill in the art that the number of dosesadministered to a patient is dependent upon the extent of the disease orcondition or the desired result, as well as the response of anindividual patient to the treatment. For example, a large tumor mayrequire more doses than a smaller tumor. A patient with diseased cardiacor skeletal muscle tissue may require more doses than a healthy athletewho desires increased muscle cell growth. In some cases, however, onepatient may require fewer doses than another patient having the samecondition, if first patient responds more favorably to the therapeuticcomposition than the other patient. Thus, it is within the scope of thepresent invention that a suitable number of doses includes any numberrequired to treat a given disease or to achieve a desired effect.

As discussed above, a therapeutic composition of the present inventionis administered to a patient in a manner effective to deliver thecomposition to a cell, a tissue, and/or systemically to the patient,whereby regulation of the prelamin A processing pathway and downstreambiological activities is achieved as a result of the administration ofthe composition. Suitable administration protocols include any in vivoor ex vivo administration protocol. The preferred routes ofadministration will be apparent to those of skill in the art, dependingon the type of condition to be prevented or treated; whether thecomposition is nucleic acid based, protein based, or cell based; and/orthe target cell/tissue. For proteins or nucleic acid molecules,preferred methods of in vivo administration include, but are not limitedto, intravenous administration, intraperitoneal administration,intramuscular administration, intranodal administration, intracoronaryadministration, intraarterial administration (e.g., into a carotidartery), subcutaneous administration, transdermal delivery,intratracheal administration, subcutaneous administration,intraarticular administration, intraventricular administration,inhalation (e.g., aerosol), intracranial, intraspinal, intraocular,intranasal, oral, bronchial, rectal, topical, vaginal, urethral,pulmonary administration, impregnation of a catheter, and directinjection into a tissue. Combinations of routes of delivery can be usedand in some instances, may enhance the therapeutic effects of thecomposition.

Ex vivo administration refers to performing part of the regulatory stepoutside of the patient, such as administering a composition (nucleicacid or protein) of the present invention to a population of cellsremoved from a patient under conditions such that the compositioncontacts and/or enters the cell, and returning the cells to the patient.Ex vivo methods are particularly suitable when the target cell caneasily be removed from and returned to the patient.

Many of the above-described routes of in vivo administration, includingintravenous, intraperitoneal, intradermal, and intramuscularadministrations can be performed using methods standard in the art.Aerosol (inhalation) delivery can also be performed using methodsstandard in the art (see, for example, Stribling et al., Proc. Natl.Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein byreference in its entirety). Oral delivery can be performed by complexinga therapeutic composition of the present invention to a carrier capableof withstanding degradation by digestive enzymes in the gut of ananimal. Examples of such carriers, include plastic capsules or tablets,such as those known in the art.

One method of local administration is by direct injection. Directinjection techniques are particularly useful for administering acomposition to a cell or tissue that is accessible by surgery, andparticularly, on or near the surface of the body. Administration of acomposition locally within the area of a target cell refers to injectingthe composition centimeters and preferably, millimeters from the targetcell or tissue.

Various methods of administration and delivery vehicles disclosed hereinhave been shown to be effective for delivery of a nucleic acid moleculeto a target cell, whereby the nucleic acid molecule transfected the celland was expressed. In many studies, successful delivery and expressionof a heterologous gene was achieved in preferred cell types and/or usingpreferred delivery vehicles and routes of administration of the presentinvention. All of the publications discussed below and elsewhere hereinwith regard to gene delivery and delivery vehicles are incorporatedherein by reference in their entirety.

For example, using liposome delivery, U.S. Pat. No. 5,705,151, issuedJan. 6, 1998, to Dow et al. demonstrated the successful in vivointravenous delivery of a nucleic acid molecule encoding a superantigenand a nucleic acid molecule encoding a cytokine in a cationic liposomedelivery vehicle, whereby the encoded proteins were expressed in tissuesof the animal, and particularly in pulmonary tissues. In addition, Liuet al., Nature Biotechnology 15:167, 1997, demonstrated that intravenousdelivery of cholesterol-containing cationic liposomes containing genespreferentially targets pulmonary tissues and effectively mediatestransfer and expression of the genes in vivo.

Several publications by Dzau and collaborators demonstrate thesuccessful in vivo delivery and expression of a gene into cells of theheart, including cardiac myocytes and fibroblasts and vascular smoothmuscle cells using both naked DNA and Hemagglutinating virus ofJapan-liposome delivery, administered by both incubation within thepericardium and infusion into a coronary artery (intracoronary delivery)(See, for example, Aoki et al., 1997, J. Mol. Cell, Cardiol. 29:949-959;Kaneda et al., 1997, Ann N.Y. Acad. Sci. 811:299-308; and von der Leyenet al., 1995, Proc Natl Acad Sci USA 92:1137-1141).

Delivery of numerous nucleic acid sequences has been accomplished byadministration of viral vectors encoding the nucleic acid sequences.Using such vectors, successful delivery and expression has been achievedusing ex vivo delivery (See, of many examples, retroviral vector; Blaeseet al., 1995, Science 270:475-480; Bordignon et al., 1995, Science270:470-475), nasal administration (CFTR-adenovirus-associated vector),intracoronary administration (adenoviral vector and Hemagglutinatingvirus of Japan, see above), intravenous administration (adeno-associatedviral vector; Koeberl et al., 1997, Proc Natl Acad Sci USA94:1426-1431). A publication by Maurice et al. (1999, J. Clin. Invest.104:21-29) demonstrated that an adenoviral vector encoding aβ2-adrenergic receptor, administered by intracoronary delivery, resultedin diffuse multichamber myocardial expression of the gene in vivo, andsubsequent significant increases in hemodynamic function and otherimproved physiological parameters. Taken together, all of the abovestudies in gene therapy indicate that delivery and expression of arecombinant nucleic acid molecule according to the present invention isfeasible.

Another method of delivery of recombinant molecules is in anon-targeting carrier (e.g., as “naked” DNA molecules, such as istaught, for example in Wolff et al., 1990, Science 247, 1465-1468). Suchrecombinant nucleic acid molecules are typically injected by direct orintramuscular administration. Recombinant nucleic acid molecules to beadministered by naked DNA administration include an isolated nucleicacid molecule of the present invention, and preferably includes arecombinant molecule of the present invention that preferably isreplication, or otherwise amplification, competent. A naked nucleic acidreagent of the present invention can comprise one or more nucleic acidmolecules of the present invention including a bicistronic recombinantmolecule. Naked nucleic acid delivery can include intramuscular,subcutaneous, intradermal, transdermal, intranasal and oral routes ofadministration, with direct injection into the target tissue (e.g.,skeletal muscle or cardiac muscle) being most preferred. A preferredsingle dose of a naked nucleic acid vaccine ranges from about 1 nanogram(ng) to about 100 μg, depending on the route of administration and/ormethod of delivery, as can be determined by those skilled in the art.Suitable delivery methods include, for example, by injection, as drops,aerosolized and/or topically. In one embodiment, pure DNA constructscover the surface of gold particles (1 to 3 μm in diameter) and arepropelled into skin cells or muscle with a “gene gun.”

In the method of the present invention, therapeutic compositions can beadministered to any member of the Vertebrate class, Mammalia, including,without limitation, primates, rodents, livestock and domestic pets.Livestock include mammals to be consumed or that produce useful products(e.g., sheep for wool production). Preferred mammals to treat using acomposition of the invention include humans, dogs, cats, mice, rats,sheep, cattle, horses and pigs, with humans being most preferred.

The discovery by the present inventor has also led the inventor topropose using this information to identify compounds that regulatemyoblast activation and differentiation through a variety of differentassays. Such methods are useful for identifying therapeutic reagents fortreating cardiac and skeletal muscle disorders and diseases and/or forpromoting myoblast activation and cardiac and skeletal muscle growth;for identifying proteins and cell lines containing specific mutationsthat will permit the elucidation of protein processing pathways relevantto normal heart and skeletal muscle development, disease development inheart and skeletal muscle, and cancer development and progression; andfor identifying additional therapeutic targets for heart disease,muscular dystrophy, and cancer prevention. For example, it is an aspectof the invention to identify genes to screen for mutations leading toheart and skeletal muscle diseases, to use the gene products of genesidentified as targets for therapeutic intervention in diseases of thecardiac and skeletal muscle.

One embodiment of the invention relates to a method to identifycompounds that regulate myoblast activation and differentiation,comprising: (a) contacting a cell that expresses a prelamin A protein ora prelamin A pre peptide with a test compound under conditions suitablefor modulation (regulation, increase or decrease, change, modification)of the activity of the prelamin A protein or prelamin A pre peptide bythe test compound; and (b) detecting modulation of the activity of theprelamin A protein or prelamin A pre peptide by the test compound.

In this embodiment, the step of detecting can include, but is notlimited to: detecting whether the test protein regulates prelamin A prepeptide transport in a cell; detecting whether the test proteinregulates the processing of prelamin A in a cell; detecting whether thetest protein regulates myoblast activation or differentiation; and/ordetecting comprises detecting binding between the prelamin A protein orprelamin A pre peptide and the test compound. In one embodiment, thestep of detecting comprises detecting an increase in myoblast activationand differentiation in the absence of correcting the prelamin Aprocessing deficiency. Each of these steps of detecting can be comparedto the various activity or parameter in the absence of the testcompound. The steps of detecting are described in more detail below.

In one embodiment, the step of detecting comprises detecting bindingbetween the prelamin A protein or prelamin A pre peptide and the testcompound. Such an assay need not be a cell based assay (e.g.,immunoprecipitation assay), although cells can be particularly usefulfor this type of assay (e.g. a yeast two hybrid system). Accordingly,one embodiment of the invention relates to a method to identifycompounds that regulate myoblast activation and differentiation,comprising: (a) contacting a prelamin A protein or a prelamin A prepeptide with a test compound under conditions suitable for binding ofthe prelamin A protein or prelamin A pre peptide by the test compound;and (b) detecting binding of the prelamin A protein or prelamin A prepeptide by the test compound.

The test compound can include a variety of different types of compounds.In one aspect, the test compound is a protein encoded by a gene that isa candidate for regulation of prelamin A processing or prelamin A prepeptide transport in the cell. For example, suitable candidates includehuman homologues of a gene in the yeast a-type mating pheromonesignaling pathway, or a gene encoding a candidate receptor for theprelamin A pre peptide. In another aspect, the test compound is apharmaceutical compound. In one embodiment, the test compound is aputative pharmaceutical compound for use in the treatment of cardiac andskeletal muscle disorders, wherein an increase in the processing ofprelamin A in the cell or an increase in myoblast activation anddifferentiation in the presence of the compound as compared to in theabsence of the compound indicates that the compound is a therapeuticcompound for use in the treatment of cardiac and skeletal muscledisorders. In yet another aspect, the test compound is a homologue of aprelamin A protein, a prelamin A pre peptide, or a prelamin A processingenzyme or downstream signal transduction molecule, or a gene encodingany of these test compounds.

Cells useful in the present assay include any cell expressing theprelamin A protein or prelamin A pre peptide, including, but not limitedto, a differentiating cardiac myocyte or a differentiating skeletalmyocyte, or a cell that has been transfected with a nucleic acidmolecule encoding the prelamin A protein or prelamin A pre peptide. Theprelamin A can be processing deficient (e.g., either a naturallyoccurring mutant or a synthetically created mutant), or the cellexpressing the prelamin A protein or prelamin A pre peptide can be aprelamin A processing deficient cell (e.g., an isolate from a patient ora laboratory created cell). The cells can include cardiac myocytes orskeletal myocytes.

For example, one embodiment of the invention relates to a method toidentify compounds that regulate myoblast activation and differentiationin a cell, comprising: (a) contacting an isolated prelamin Aprocessing-deficient cell with a test compound for regulation ofmyoblast activation and differentiation; (b) contacting the isolatedcell with test compound for regulation of myoblast activation anddifferentiation; and (c) detecting whether the test compound regulatesan activity in the cell selected from the group consisting of: prelaminA processing, prelamin A pre peptide transport, and myoblast activationor differentiation, as compared to in the absence of the test compound.The present inventor has identified disease mutations that inhibitproper prelamin A processing in myocyte cell lines. This protein, andthese transfected cell lines, will permit the elucidation of the enzymesand steps in the prelamin A processing pathway by complementationexperiments. These cell lines will also serve as a reagent to testtherapeutic agents to rectify the prelamin A processing deficiencies.Cell lines generated from a patient identified as carrying this laminA/C mutation can be used for similar purposes.

Having generally described various methods of identification of theinvention, more particular details of the assays that apply to one ormore of the methods above will now be described. For example, it will beapparent that the methods described above are typically cell-basedassays, but may include cell-free assays, such as when one wishes toassess binding of one protein to another.

In one aspect of these methods, the methods can include a step ofcontacting a cell that expresses a prelamin A protein or prelamin A prepeptide (including a prelamin A processing deficient cell), orcontacting a prelamin A protein or a prelamin A pre peptide directly,with a putative regulatory compound (a test compound, including a gene,protein or candidate drug), followed by a step of detecting an effect onthe cell or protein, preferably as compared to in the absence of theputative regulatory compound.

In these embodiments, a change in the regulation of some aspect of theprelamin A processing pathway, including downstream events that resultfrom activation of this pathway, in the presence of the test compound ascompared to in the absence of the test compound indicates that the testcompound is an regulator of the prelamin A processing pathway. If theinitial assay is not a cell-based assay (e.g., detects only binding ofthe test compound to a protein such as prelamin A), then the compoundcan be further tested, if desired, in a cell-based assay to determinewhether the compound inhibits or enhances a biological activity withinthe prelamin A processing pathway. Such further steps will help detectthe mode of action of the compound and whether it might be an agonist orantagonist of the prelamin A processing pathway.

As used herein, the term “putative” or “test” or “candidate” refers tocompounds having an unknown or previously unappreciated regulatoryactivity in a particular process. As such, the term “identify” isintended to include all compounds, the usefulness of which as aregulatory compound according to the invention determined by a method ofthe present invention.

The methods of the present invention include contacting test compoundsand cells, proteins or genes with one another to detect binding of onecomponent to another or to detect the effect of the contact onexpression and/or biological activity of one or more of the components.The step of contacting can be performed by any suitable method,depending on how the test compound and the cell, proteins, or genes areprovided. For example, cells expressing prelamin A or a prelamin A prepeptide can be grown in liquid culture medium or grown on solid mediumin which the liquid medium or the solid medium contains the compound tobe tested. In addition, as described above, the liquid or solid mediumcontains components necessary for cell growth, such as assimilablecarbon, nitrogen and micro-nutrients. Cell lysates can be combined withother cell lysates and/or the compound to be tested in any suitablemedium. In another embodiment, proteins and/or cell lysates containingsuch proteins can be immobilized on a substrate such as: artificialmembranes, organic supports, biopolymer supports and inorganic supports.The protein can be immobilized on the solid support by a variety ofmethods including adsorption, cross-linking (including covalentbonding), and entrapment. Adsorption can be through van del Waal'sforces, hydrogen bonding, ionic bonding, or hydrophobic binding.Exemplary solid supports for adsorption immobilization include polymericadsorbents and ion-exchange resins. Solid supports can be in anysuitable form, including in a bead form, plate form, or well form. Theputative regulatory compound can be contacted with the immobilizedprotein by any suitable method, such as by flowing a liquid containingthe compound over the immobilized protein.

The present methods involve contacting cells or proteins with thecompound being tested for a sufficient time to allow for interactionwith the cell or protein, and regulation of the cell by the compound.The period of contact with the compound being tested can be varieddepending on the result being measured, and can be determined by one ofskill in the art. For example, for binding assays, a shorter time ofcontact with the compound being tested is typically suitable, than whenactivation is assessed. As used herein, the term “contact period” refersto the time period during which the proteins are in contact with thecompound being tested and/or the time period during which the proteinsor cells and the test compounds are in contact (or in a condition wherecontact is possible) with each other. The term “incubation period”refers to the entire time during which, for example, cells are allowedto grow prior to evaluation, and can be inclusive of the contact period.Thus, the incubation period includes all of the contact period and mayinclude a further time period during which the compound being tested isnot present but during which growth is continuing (in the case of a cellbased assay) prior to scoring. It will be recognized that shorterincubation times are preferable because compounds can be more rapidlyscreened.

The conditions under which a cell or cell lysate is contacted with aputative regulatory compound, such as by mixing, are any suitableculture or assay conditions and includes an effective medium in whichthe cell can be cultured or in which the cell lysate can be evaluated inthe presence and absence of a putative regulatory compound. Similarly,the conditions under which proteins (e.g., prelamin A or pre peptide)are contacted with a putative regulatory compound are any suitable assayconditions, such as by immobilization of the protein or peptide on asubstrate in conditions under which the protein or peptide can contactthe putative regulatory compound.

Cells of the present invention can be cultured in a variety ofcontainers including, but not limited to, tissue culture flasks, testtubes, microtiter dishes, and petri plates. Culturing is carried out ata temperature, pH and carbon dioxide content appropriate for the cell.Such culturing conditions are also within the skill in the art.Acceptable protocols to contact a cell with a putative regulatorycompound in an effective manner include the number of cells percontainer contacted, the concentration of putative regulatorycompound(s) administered to a cell, the incubation time of the putativeregulatory compound with the cell, and the concentration of compoundadministered to a cell. Determination of such protocols can beaccomplished by those skilled in the art based on variables such as thesize of the container, the volume of liquid in the container, the typeof cell being tested and the chemical composition of the putativeregulatory compound (i.e., size, charge etc.) being tested. A preferredamount of putative regulatory compound(s) comprises between about 1 nMto about 10 mM of putative regulatory compound(s) per well of a 96-wellplate.

Suitable cells for use with the present invention include any cell thatendogenously expresses prelamin A or prelamin A pre peptide (wild-typeor processing-deficient), or which has been transfected with andexpresses a recombinant prelamin A or prelamin A pre peptide asdisclosed herein. Such cells can include cells with normal prelamin Aprocessing and prelamin A processing-deficient cells (which may containnormal prelamin A). In one embodiment, host cells genetically engineeredto express a prelamin A or pre peptide can be used as an endpoint in theassay; e.g., as measured by a chemical, physiological, biological, orphenotypic change, induction of a host cell gene or a reporter gene,change in cAMP levels, adenylyl cyclase activity, host cell G proteinactivity, host cell kinase activity, proliferation, differentiation,etc. Cells for use with the present invention include mammalian,invertebrate, plant, insect, fungal, yeast and bacterial cells.Preferred cells include mammalian cells. Other preferred cells includecardiac or skeletal muscle myocytes and preferably, differentiatingcardiac or skeletal muscle myocytes. Prelamin A processing-deficientcells useful in the present invention have been isolated by the presentinventor are described in the Examples section.

As discussed above, the step of detecting whether a test compound bindsto prelamin A or pre peptide (or another protein or gene in a cell) orregulates a parameter of prelamin A processing and/or its downstreambiological effects, can be performed by any suitable method. Suchmethods include, but are not limited to: measurement of protein-proteinbinding or interaction, measurement of transcription of prelamin A,measurement of translation of prelamin A, measurement ofposttranslational modification of prelamin A, measurement of properprocessing of the pre peptide, measurement of pre peptide signaltransduction, measurement of lamin A incorporation into the nuclearlamina structure, measurement of transcriptional regulation ofmuscle-specific genes and/or cell cycle arrest, measurement of nuclearlamina morphology, measurement of pre peptide transport, measurement oflamin A localization, measurement of myocyte cell fusion, and/ormeasurement of myoblast activation and differentiation. Techniques forperforming such measurements are known in the art, and include a varietyof binding assays, western blotting, immunocytochemistry, flowcytometry, other immunological based assays, phosphorylation assays,kinase assays, immunofluorescence microscopy, RNA assays,immunoprecipitation, cytokine assays, evaluation of cell morphology, insitu hybridization, and other biological assays. Binding assays includeBIAcore machine assays, immunoassays such as enzyme linkedimmunoabsorbent assays (ELISA) and radioimmunoassays (RIA), ordetermination of binding by monitoring the change in the spectroscopicor optical properties of the proteins through fluorescence, UVabsorption, circular dichroism, or nuclear magnetic resonance (NMR).Binding and/or interaction between two proteins can be determined usingyeast two hybrid systems. Methods for evaluating prelamin A processingand its biological effects are described in the Examples section.

As discussed above, in vitro cell based assays may be designed to screenfor compounds that regulate prelamin A processing and associatedbiological events at either the transcriptional or translational level.For example, one embodiment of the invention relates to a method toidentify a genes or gene products that regulate the processing ofprelamin A or activities downstream of the prelamin A processing and prepeptide signal transduction. In one aspect, a nucleic acid sequenceencoding a reporter molecule can be linked to a regulatory element ofprelamin A or an associated protein and used in appropriate intactcells, cell extracts or lysates to identify compounds that modulateprelamin A gene expression or expression of a gene involved in prelaminA processing or pre peptide signal transduction. Appropriate cells orcell extracts can be prepared, if desired, from any cell type thatnormally expresses a gene encoding prelamin A, thereby ensuring that thecell extracts contain the transcription factors required for in vitro orin vivo transcription. The screen can be used to identify compounds thatmodulate the expression of the reporter construct. In such screens, thelevel of reporter gene expression is determined in the presence of thetest compound and compared to the level of expression in the absence ofthe test compound.

The method can also include the step of detecting the expression of atleast one, and preferably more than one, of the downstream genes thatare regulated by prelamin A processing and the release of the prepeptide, or of the genes that are involved in the processing of prelaminA. As used herein, the term “expression”, when used in connection withdetecting the expression of a downstream gene of the present invention,can refer to detecting transcription of the gene and/or to detectingtranslation of the gene. To detect expression of a gene refers to theact of actively determining whether a gene is expressed or not. This caninclude determining whether the gene expression is upregulated ascompared to a control, downregulated as compared to a control, orunchanged as compared to a control. Therefore, the step of detectingexpression does not require that expression of the gene actually isupregulated or downregulated, but rather, can also include detectingthat the expression of the gene has not changed (i.e., detecting noexpression of the gene or no change in expression of the gene).Expression of transcripts and/or proteins is measured by any of avariety of known methods in the art. For RNA expression, methods includebut are not limited to: extraction of cellular mRNA and northernblotting using labeled probes that hybridize to transcripts encoding allor part of one or more of the genes of this invention; amplification ofmRNA expressed from one or more genes using gene-specific primers, ifavailable and reverse transcriptase—polymerase chain reaction, followedby quantitative detection of the product by any of a variety of means;extraction of total RNA from the cells, which is then labeled and usedto probe cDNAs or oligonucleotides encoding all or part of the genes ofthis invention, arrayed on any of a variety of surfaces. The term“quantifying” or “quantitating” when used in the context of quantifyingtranscription levels of a gene can refer to absolute or to relativequantification. Absolute quantification may be accomplished by inclusionof known concentration(s) of one or more target nucleic acids andreferencing the hybridization intensity of unknowns with the knowntarget nucleic acids (e.g. through generation of a standard curve).Alternatively, relative quantification can be accomplished by comparisonof hybridization signals between two or more genes, or between two ormore treatments to quantify the changes in hybridization intensity and,by implication, transcription level.

Another embodiment of the methods described above includes identifyingwhether a candidate gene is a gene that encodes a product that isinvolved in either prelamin A processing, or downstream activitiesresulting from prelamin A processing and the release of the pre peptide.Such methods are typically performed by protein-protein interactionassays to identify gene products that interact with a given protein(e.g., prelamin A or pre peptide), or by complementation assays usingcell lines expressing various proteins in the pathway.

In one embodiment, the invention includes a method to identify humangenes that regulate myoblast activation and differentiation, comprising:(a) contacting a probe with a source of human DNA from heart or skeletalmuscle tissue under low stringency conditions, wherein the probe is anucleic acid sequence from a gene in the yeast a-type mating pheromonesignal transduction pathway; (b) identifying genes in the source ofhuman DNA that hybridize to the probe; and (c) detecting whether genesthat hybridize to the probe encode a protein that corrects a prelamin Aprocessing deficiency or that increases myoblast activation anddifferentiation. For example, the gene in the yeast a-type matingpheromone signal transduction pathway can include a gene that isassociated with a biological function selected from: transcriptionalactivation of pheromone responsive genes, post-transcriptional blockadeof the cell cycle, and cell fusion pathway activation. As discussedabove, the only protein known to be post-translationally processed inthe same manner as prelamin A is the yeast a-type mating pheromone.Yeast a-factor (Mat a) peptide activates a receptor-coupled G proteinwhich, in turn activates a mitogen-activated protein (MAP) kinase. Thissplits the signal into three branches: transcriptional activation ofpheromone responsive genes, post-transcriptional blockade of the cellcycle, and cell fusion pathway activation. Therefore, human homologuesof genes in the yeast a-type mating pheromone pathway are goodcandidates for other genes relevant to the prelamin A processing and prepeptide signal transduction, including candidates for genes that containmutations that cause heart and skeletal muscle diseases. These genes andtheir protein products are putative targets for therapies to preventcardiac and skeletal muscle diseases.

Methods of creating probes suitable for use in a homologue screen areknown in the art and include the preparation of degenerate primers.Methods for identifying genes using hybridization are well known in theart. It is generally recognized that nucleic acids are denatured byincreasing the temperature or decreasing the salt concentration of thebuffer containing the nucleic acids. Under low stringency conditions(e.g., low temperature and/or high salt) hybrid duplexes (e.g., DNA:DNA,RNA:RNA, or RNA:DNA) will form even where the annealed sequences are notperfectly complementary. Thus specificity of hybridization is reduced atlower stringency. Conversely, at higher stringency (e.g., highertemperature or lower salt) successful hybridization requires fewermismatches. Hybridization conditions are described in detail above.

Yet another embodiment of the invention relates to a method to identifyan inhibitor of prelamin A farnesylation, comprising the steps of: (a)contacting an isolated cell that expresses prelamin A with a putativeregulator of prelamin A farnesylation; and (b) detecting whetherfarnesylation of prelamin A is inhibited by the putative regulator. Thefarnesylation and subsequent processing of pre-lamin A is essential forits proper function in cardiac and skeletal myocyte formation.Activation of the Ras oncogene is responsible for greater than 70% ofcolorectal cancer cases. Farnesylation of Ras is the initial step in Rasactivation. Currently, farnesylation inhibitors are being used inclinical trials to prevent Ras activation and cancer progression.Therefore, identification of the enzymatic steps in the prelamin Afarnesylation pathway will identify putative targets for additionaltherapies to prevent Ras activation and colorectal cancer. In addition,the present inventor has found that inhibition of farnesylation usingknown inhibitors in differentiating muscle cells causes muscle celldegeneration and muscle cell death. Based on the present inventor'sdiscovery, one can now see that inhibition of proper prelamin Aprocessing is most likely the cause of this result. Therefore, thepresent inventor proposes using inhibitors of prelamin A processing, andparticularly, farnesylation inhibitors, to inhibit or treat muscle cellcancers via inhibition of the prelamin A processing pathway.

In the method of identification of farnesylation inhibitors, the cellcan be any suitable cell expressing prelamin A as discussed previouslyherein. In one embodiment, the cell is selected from the groupconsisting of a differentiating cardiac myocyte and a differentiatingskeletal myocyte. In another embodiment, the cell has been transfectedwith a nucleic acid molecule encoding prelamin A. The step of detectingincludes detecting whether prelamin A farnesylation is reduced ascompared to in the absence of the putative inhibitor compound. Inadditional steps, one can detect whether inhibitors of prelamin Afarnesylation detected in step (b) regulate prelamin A processing in thecell, wherein detection of reduced prelamin A processing in the presenceof the regulator indicates that the regulator may be useful fortreatment of muscle cell cancers. Alternatively, one can detect whetherinhibitors of prelamin A farnesylation detected in step (b) causemyoblast dissociation or myoblast cell death, wherein detection ofincreased myoblast dissociation or myoblast cell death in the presenceof the regulator indicates that the regulator may be useful fortreatment of muscle cell cancers. Methods of contacting and detectinguseful in this aspect of the invention have been described generallyabove for other cell-based assays and are additionally known in the art.Methods of detecting farnesylation of a protein are described, forexample, in: Proc Natl. Acad. Sci. 89:3000-3004 (1992); or Proc. Natl.Acad. Sci. 97:11626-11631 (2000), incorporated herein by reference intheir entireties. Methods of detecting myoblast dissociation or celldeath are also well known in the art.

Accordingly, another embodiment of the invention is a method to treat amuscle cell cancer, comprising administering to a patient with a musclecell cancer or metastatic cancer thereof a compound that inhibitsprelamin A processing and myoblast differentiation. The compound caninclude any known or as yet unknown compound that will inhibit prelaminA processing and particularly, farnesylation of prelamin A. Oneparticularly preferred compound can include a statin that is toxic tomyocytes. Methods to identify such compounds have been describedpreviously herein. Muscle cell cancers to treat using this methodinclude, but are not limited to: myosarcoma, myeloma, myoma,rhabdomyosarcoma, and malignant uterine fibroids.

Another embodiment of the present invention relates to a method todiagnose a disorder associated with prelamin A processing defects, or toscreen for patients that carry mutations that may cause cardiac orskeletal muscle disorders. The method includes the steps of detectingexpression or biological activity of prelamin A or the prelamin A prepeptide in a tissue of a patient suspected of having a disorder, andcomparing the expression or biological activity to a control, wherein adifference in the expression or biological activity of prelamin A or thepre peptide (including a gene encoding such proteins) in a tissue of thepatient as compared to the control indicates a positive diagnosis of adisorder associated with improper prelamin A processing. Once additionalenzymes and proteins in the processing and signal transduction pathwayhave been elucidated, these proteins and genes encoding them can also beused to diagnose or screen patients for a cardiac or skeletal muscledisorder. The terms “diagnose”, “diagnosis”, “diagnosing” and variantsthereof refer to the identification of a disease or condition on thebasis of its signs and symptoms. As used herein, a “positive diagnosis”indicates that the disease or condition, or a potential for developingthe disease or condition, has been identified. In contrast, a “negativediagnosis” indicates that the disease or condition, or a potential fordeveloping the disease or condition, has not been identified.

According to the present invention, the term “cell sample” can be usedgenerally to refer to a sample of any type which contains cells to beevaluated by the present method, including but not limited to, a sampleof isolated cells, a tissue sample and/or a bodily fluid sample.According to the present invention, a sample of isolated cells is aspecimen of cells, typically in suspension or separated from connectivetissue which may have connected the cells within a tissue in vivo, whichhave been collected from an organ, tissue or fluid by any suitablemethod which results in the collection of a suitable number of cells forevaluation by the method of the present invention. The cells in the cellsample are not necessarily of the same type, although purificationmethods can be used to enrich for the type of cells which are preferablyevaluated. Cells can be obtained, for example, by scraping of a tissue,processing of a tissue sample to release individual cells, or isolationfrom a bodily fluid. A tissue sample, although similar to a sample ofisolated cells, is defined herein as a section of an organ or tissue ofthe body which typically includes several cell types and/or cytoskeletalstructure which holds the cells together. One of skill in the art willappreciate that the term “tissue sample” may be used, in some instances,interchangeably with a “cell sample”, although it is preferably used todesignate a more complex structure than a cell sample. A tissue samplecan be obtained by a biopsy, for example, including by cutting, slicing,or a punch. A bodily fluid sample, like the tissue sample, contains thecells to be evaluated, and is a fluid obtained by any method suitablefor the particular bodily fluid to be sampled. Nucleic acids can beprepared from any of these samples for screening.

Methods suitable for detecting transcription or translation have beendescribed elsewhere herein and include any suitable method for detectingand/or measuring mRNA levels or protein levels from a cell, cell extractor tissue. Methods for detecting transcription include, but are notlimited to: polymerase chain reaction (PCR), reverse transcriptase PCR(RT-PCR), in situ hybridization, Northern blot, sequence analysis, anddetection of a reporter gene. Such methods for detection oftranscription levels are well known in the art, and many of such methodsare described in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Labs Press, 1989 and/or in Glick et al.,Molecular Biotechnology: Principles and Applications of Recombinant DNA,ASM Press, 1998; Sambrook et al., ibid., and Glick et al., ibid. areincorporated by reference herein in their entireties. Methods suitablefor the detection of protein include, but are not limited to, immunoblot(e.g., Western blot), enzyme-linked immunosorbant assay (ELISA),radioimmunoassay (RIA), immunoprecipitation, immunohistochemistry andimmunofluorescence. Such methods are well known in the art.

Typically, in a diagnostic or screening assay, a test level ofexpression of a protein or nucleic acid is compared to a baseline level,or control. According to the present invention, a “baseline level” is acontrol level, and in some embodiments, a normal level of protein orgene expression or activity against which a test level of protein orgene expression or biological activity (i.e., in the patient sample) canbe compared. The method for establishing a baseline level of protein orgene expression or activity is selected based on the sample type, thetissue or organ from which the sample is obtained, the status of thepatient to be evaluated, and the focus or goal of the assay (e.g.,diagnosis, staging, monitoring). Preferably, the method is the samemethod that will be used to evaluate the sample in the patient. Baselinelevels can be established using an autologous control sample obtainedfrom the patient, an autologous level in a previous sample from the samepatient, or using control samples that were obtained from a populationof matched individuals.

The following examples are provided for the purpose of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Materials and Methods

Plasmids. The wild type prelamin A cDNA, a gift from M. Sinensky(Eastern Tennessee State University), was sub-cloned from pMMLA intopAlter-1 (Promega). Oligonucleotide primers were designed containing sixdifferent lamin A/C sequence mutations that cause DCM; C178G (Arg60Gly),T254G (Leu85Arg), C585G (Asn195Lys), A608G (Glu203Gly), G266T(Arg89Leu), and G1130A (Arg377His). Each primer contains 10-11 bp offlanking sequence homology on either side of the mutation. Site directedmutagenesis was performed on the wild type prelamin A cDNA/pAlter-1construct using Altered Sites (Promega). DNA was isolated fromindividual transformants and sequenced to identify clones containing theinduced mutations.

The wild type and six mutant prelamin A/pAlter-1 plasmid DNAs were usedas templates in PCR reactions with primers designed to permitsub-cloning of the prelamin A cDNA PCR products in-frame with theN-terminal GFP coding sequence of pEGFP-C1 (Clonetech). The prelamin AcDNA inserts were sequenced in their entirety to confirm the presence ofthe desired mutation and the absence of any spurious Taqpolymerase-induced mutations.

Cells and Transfections. C2C12 and H9C2 cells were incubated for 24 h inDMEM containing 10% (vol/vol) fetal bovine serum. Effectene (Qiagen) wasused to transfect plasmid DNA into cells following the manufacturersinstructions. Non-differentiating cells were allowed to continue growingfor an additional 24 h before being processed for microscopy, or untilnear confluent (2 to 3 days) for protein isolation. For differentiation,H9C2 and C2C12 cells were allowed to continue growing 36 hours aftertransfection prior to incubation in DMEM containing 2% (vol/vol) horseserum. In the case of H9C2 cells, differentiation media also contained10 nM retinoic acid. C2C12 cells were differentiated for 48 hours or 8days, and H9C2 cells were differentiated 7 days prior to processing formicroscopy.

Indirect immunofluorescence (IF). All slides were fixed by treating withcold 70% methanol/30% acetone solution for 10 min. Immunostaining wasperformed by treating fixed cells with anti-lamin A/C mouse monoclonalantibody (NCL-LAM-A/C)(1:50) (Novocastra Laboratories), or anti-desminrabbit polyclonal antibody (1:50) (Sigma). The secondary antibody usedwith the anti-lamin A/C antibody was Texas red-conjugated horseanti-mouse (1:300) (Vector Laboratories), and the secondary antibodyused with the anti-desmin antibody was Texas red-conjugated goatanti-rabbit (1:300) (Vector Laboratories). Cover slips were mounted withVectashield containing DAPI (Vector Laboratories).

Western blots. Non-differentiated C2C12 cells were lysed in cell buffer(1% Triton X-100, 20 mM Tris-Cl, 7.5, 10 mM NaCl, 5 mM MgCl₂) containing46 μg/ml Leupeptin (Sigma), 10 μg/ml Aprotinin (Sigma), and 250 μg/mlAE-BSF (Sigma) for 10 min on ice. Total protein concentration wasdetermined using BioRad Protein Assay reagent. One volume of 2× Laemliloading buffer was added and the samples were boiled for 2 min. Proteinswere separated by polyacrylamide gel electrophoresis and transferred toinmobilon-P (Millipore). Western blots were probed with an anti-GFPrabbit polyclonal antibody (SC-8334) (1:1000) (Santa Cruz Biotechnology)and HRP-conjugated goat anti-rabbit secondary antibody (1:5000)(BioRad), or an anti-prelamin A goat polyclonal antibody (SC-6214)(1:100) (Santa Cruz Biotechnology) and HRP-conjugated mouse anti-goatsecondary antibody (1:500) (Santa Cruz Biotechnology). Secondaryantibody detection was performed using Super signal HRP (Pierce).

Example 1

The following example demonstrates that mutations that cause dilatedcardiomyopathy result in aberrant lamin A localization and laminaformation.

Indirect IF microscopic studies of peptide-tagged lamin A proteinscontaining disease-causing mutations have demonstrated that thesemutations can affect the structure of the nuclear lamina. However,changes in the structure of the nuclear lamina can mask antigenic sitesand inhibit antibody access. Consequently, it is unclear if the changesin lamina structure revealed by indirect IF reflect the truelocalization of, and structures formed by, the mutant lamin A proteins,or alterations in the interactions between the antibodies used in theseexperiments and the mutant lamin A proteins. In order to directlyvisualize the effects of disease-causing mutations on lamina formationand lamin A localization, the present inventor created a prelamin Afusion protein expression construct in which the coding sequence for GFPwas fused to the N-terminal end of the prelamin A cDNA. An N-terminalGFP-prelamin A fusion protein has been shown to be processed andfunction normally in mammalian cells.

Six different single nucleotide substitutions that cause DCM wereindependently introduced into the GFP prelamin A cDNA by site-directedmutagenesis. These mutations result in the amino acid substitutions(relative to SEQ ID NO:4) Arg60Gly, Leu85Arg, Asn195Lys, Glu203Gly,Arg89Leu and Arg377His. The wild type and mutant expression constructswere transfected into mouse C2C12 skeletal myoblasts. Expression of themutant lamin proteins in C2C12 cells, which express endogenous wild typelamins A and C, approximates the heterozygous state of lamin expressionin the tissues of patients affected by these autosomal dominant diseasemutations. In order to compare the results obtained by directfluorescence microscopy of the GFP signal with those produced byindirect IF microscopy, the cells were fixed and stained with ahuman-specific anti-lamin A/C antibody. As expected, the wild typeprelamin A fusion protein incorporated into the nuclear lamina asevidenced by the more intense signal seen around the circumference ofthe nucleus (data not shown). In addition, a number of intranuclearlamin A filaments were observed as previously described.

The nuclear lamina formed in cells expressing the fusion proteinscontaining the mutations Arg60Gly, Leu85Arg and Arg377His (data notshown) are indistinguishable from those formed by the wild typeconstruct. However, extra-nuclear GFP fusion protein aggregates are seenin the transfectants expressing the Arg60Gly and Leu85Arg mutations.While the fusion protein containing the Glu203Gly mutation incorporatesefficiently into the nuclear lamina (data not shown), nuclei oftransfectants expressing this mutant protein typically contain largeareas of diffuse GFP signal that are devoid of the fine filamentousstructures seen in the wild type control. These transfectants alsocontain cytoplasmic GFP fusion protein aggregates. The expression offusion proteins containing the Asn195Lys (data not shown) and Arg89Leu(data not shown) mutations results in the formation of intranuclearlamin aggregates. All aggregates are located at the nuclear periphery asdetermined by three-dimensional volume projections of the digitallydeconvolved images (data not shown).

Increasing the gain of the GFP channel revealed nucleoplasmic GFP signalin the Arg89Leu transfectants, but not in the Asn195Lys transfectants(data not shown).

Indirect IF analysis of transfectants expressing the wild type prelaminA construct accurately represents the localization of the GFP fusionprotein, and its incorporation into the nuclear lamina and intranuclearfilaments. However, artifacts in the form of intranuclear antibodyaggregates that do not co-localize with the GFP signal are observed intransfectants containing the wild type, and each of the mutant fusionprotein constructs (data not shown). Indirect IF analysis oftransfectants expressing the Arg60Gly, Leu85Arg and Arg377His proteinconstructs demonstrates that these mutations are affecting the laminastructure such that the antibody under represents the incorporation ofthe GFP fusion proteins into the lamina, and over represents thenucleoplasmic localization of these proteins. Indirect IF analysis oftransfectants expressing fusion proteins containing the Asn195Lys,Glu203Gly and Arg89Leu mutations accurately depicts the localization ofthe mutant fusion proteins. However, antibody access to the interior ofthe intranuclear aggregates in cells expressing the Asn195Lys andArg89Leu mutations is prevented, resulting in the false representationof these mutant proteins forming hollow, ring-like structures.

Example 2

The following example demonstrates that lamin A/C mutations affectdifferent steps in prelamin A processing.

Prelamin A is processed in a sequential series of post-translationmodifications shared by the S. cerevisiae a-type mating pheromone. Todetermine if the mutations associated with DCM affect prelamin Aprocessing, equal amounts of total protein extracts from C2C12 myoblaststransfected with the wild type and mutant fusion protein constructs wereanalyzed by Western blot analysis with an anti-GFP antibody. Referringto FIG. 1, total protein was isolated from C2C12 cells that wereuntransfected (lane 1), or transfected with the wild type prelamin Afusion protein construct (lane 2), and with the prelamin A constructscontaining the Arg60Gly (lane 3), Leu85Arg (lane 4), Asn195Lys (lane 5),Glu203Gly (lane 6), Arg89Leu (lane 7), and Arg377His (lane 8) mutations.Proteins were resolved by 10% SDS/PAGE, transferred to membranes, andprobed with an anti-GFP antibody (A) or prelamin A-specific antibody(B). The migration of MW standards are indicated in kDa at left.

A protein band of the size expected for lamin A (74 kDa) fused to GFP(27 kDa) was detected in all transfectants (FIG. 1A, lanes 2-8), and wasabsent from the untransfected control (FIG. 1A, lane 1). The intensityof this band varies between samples, reflecting that the proteins wereisolated from transient transfections that resulted in differenttransfection efficiencies.

The GFP fusion protein containing the Arg89Leu mutation (FIG. 1A, lane7) has reduced mobility as compared with the other protein constructs.This protein also migrates slower on Western blots of total proteinisolated from H9C2 rat cardiac myoblast transfectants probed with theanti-GFP antibody, and from HeLa cell transfectants probed with ananti-lamin A/C antibody (data not shown). In order to confirm theArg89Leu mutation is affecting prelamin A processing, Western blotanalysis of the C2C12 protein extracts was performed using an antibodywhich specifically recognizes the C-terminal “pre” peptide of prelaminA. prelamin A was detected in the protein extracts from transfectantsexpressing the Arg89Leu mutation (FIG. 1B, lane 7), and in extracts fromcells expressing the Glu203Gly and Arg377His mutations as well (FIG. 1B,lanes 7 and 8, respectively). Furthermore, the prelamin A proteincontaining the Glu203Gly mutation has a greater mobility than thosecontaining the Arg89Leu and Arg377His mutations, demonstrating that theGlu203Gly mutation affects a different prelamin A processing step.

Example 3

The following example demonstrates that lamin A/C mutations affectmyoblast differentiation.

The observations that DCM resulting from lamin A/C gene mutations is nota congenic disorder, and that lamin A/C knockout mice fail to showskeletal and cardiac pathologies until 3-4 weeks post natal, indicatethat lamin A/C disease mutations affect tissue growth and repair, notorganogenesis. In adult skeletal muscle, mononucleated stem cellsreferred to as “satellite cells” are responsible for the regeneration ofmuscle fibers in response to injury. To test the hypothesis that laminA/C gene mutations responsible for DCM affect satellite cell function,mouse C2C12 cells transfected with the wild type and mutant prelamin Afusion protein constructs were induced to differentiate, and examinedfor defects in myocyte morphology and intercellular organization aftertwo days of growth in differentiation media. Direct fluorescencemicroscopy was used to identify myocytes expressing the GFP fusionprotein constructs. Indirect IF microscopy was performed using ananti-desmin antibody, as desmin is an early marker of myocytedifferentiation expressed throughout the cytoplasm of the myotube.

Analysis of myocytes expressing the fusion protein constructs revealsthat lamin A is not restricted to nuclear domains, and is present ineither all or none of the C2C12 myotube nuclei (data not shown). Thetransfer of lamin A between nuclei occurs at an early stage of myoblastfusion, as it was not possible to detect any myocytes containing 2 or 3nuclei in which only one or two of the nuclei contained GFP-lamin A.Myocyte morphology and intercellular organization are not affected byexpression of the wild type construct, as the nuclei within the myotubeare arranged in a normal, linear fashion (data not shown), and adjacentmyotubes are organized in a parallel rows (data not shown).

Expression of fusion proteins containing the Asn195Lys, Glu203Gly, andArg89Leu mutations results in aberrant myocyte morphology, both inmyotubes expressing these mutant proteins, and in adjacent myotubes thatdo not express the fusion proteins. Myotubes expressing these mutantproteins are thick, and characterized by angular and branchedprotrusions. Myotubes expressing the Asn195Lys and Glu203Gly proteinconstructs often contain parallel rows of nuclei (data not shown).Similar aberrant morphologies can be observed in the adjacent myotubes,which do not express the mutant fusion proteins. The expression of thesemutant proteins also affects intercellular organization as the myotubesare arranged at various angles of up to 90 degrees relative to adjacentmyotubes, and frequently grow across each other. When myotubesexpressing the Glu203Gly and Arg89Leu mutations were allowed todifferentiate for 8 days prior to processing, these effects onmorphology and cellular organization were even more pronounced.

Expression of the GFP fusion protein containing the Arg377His mutationhad a different effect on myotube morphology. Myotubes expressing thisconstruct have a very segmented appearance. Furthermore, while thenuclei are clearly defined by indirect IF with the anti-desmin antibodyin myotubes expressing the wild type and other mutant proteins, thenuclei in myotubes expressing the Arg377His mutation are poorly defined,or not at all evident (data not shown). Again, these effects are seennot only in myotubes expressing the Arg377His mutation, but also inadjacent myotubes that do not express the mutant fusion protein. Thechanges in appearance that are observed suggest that the nuclei in thesemyotubes are not membrane associated as in normal skeletal muscle, butinternalized like a subset of the nuclei in skeletal muscle fibers fromsome patients with DCM.

In order to assess the effects of mutant prelamin A expression oncardiac myoblast differentiation, H9C2 myoblasts derived from neonatalrat heart were transfected with the prelamin A GFP fusion proteinconstructs, and induced to differentiate in the presence of retinoicacid. Exposure to retinoic acid causes H9C2 cells to differentiatetowards a cardiac phenotype, which is characterized by parallel rows ofnuclei as opposed to the linear arrangement observed in skeletalmyotubes. In contrast to the distribution of the GFP fusion proteinsseen in skeletal myotubes, the fusion protein constructs are partiallylimited to nuclear domains in differentiating H9C2 myotubes (data notshown). The number of nuclei containing the GFP fusion proteins in thesemyocytes ranges from 1 to 4, with the greatest number of myotubescontaining two nuclei labeled by GFP. As normal cardiac myocytes containtwo nuclei, these results may indicate that only a subset of nuclei inthe H9C2 myotubes are active.

Expression of the fusion proteins containing the Arg60Gly and Leu85Argmutations results in the formation of myotubes with mixed cardiac andskeletal morphologies (data not shown). These myotubes contain globularareas with nuclei in parallel rows, as well as projections that appearsimilar to skeletal myotubes containing linearly arranged nuclei.Expression of the protein constructs containing the Asn195Lys andArg89Leu mutations results in the formation of aberrant, large randomlyshaped myotubes (data not shown). In contrast, the Glu203Gly mutationappears to hinder myotube formation (data not shown). The nuclei inmyotubes expressing the Glu203Gly fusion protein are typically fartherremoved from one another than in myotubes containing the wild typeprelamin A fusion protein and other mutant constructs. In addition, theindividual myoblast cell bodies are still evident and appear to betrapped in the early stages of intercellular fusion.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing Claims.

1-45. (canceled)
 46. An isolated peptide selected from the groupconsisting of: a) a peptide consisting of an amino acid sequence that isat least 70% identical to an amino acid sequence selected from the groupconsisting of: SEQ ID NO:2, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 andSEQ ID NO:19, wherein the peptide promotes myoblast differentiation; b)a peptide consisting of an amino acid sequence that differs from SEQ IDNO:2 by at least one substitution, deletion or insertion of an aminoacid residue at a position of SEQ ID NO:2 selected from the groupconsisting of: 1, 2, 5, 6, 9, 10, 11, 12, 13 and 14, wherein the peptidepromotes myoblast differentiation; c) a peptide consisting of an aminoacid sequence that differs from SEQ ID NO:16 by at least onesubstitution, deletion or insertion of an amino acid residue at aposition of SEQ ID NO:16 selected from the group consisting of: 1, 2, 5,6, 9, 10, 11, 12, 13 and 14, wherein the peptide promotes myoblastdifferentiation; d) a peptide consisting of an amino acid sequence thatdiffers from SEQ ID NO:17 by at least one substitution, deletion orinsertion of an amino acid residue at a position of SEQ ID NO:17selected from the group consisting of: 1, 2, 5, 6, 9, 10, 11, 12, and16, wherein the peptide promotes myoblast differentiation; e) a peptideconsisting of an amino acid sequence that differs from SEQ ID NO:18 byat least one substitution, deletion or insertion of an amino acidresidue at a position of SEQ ID NO:18 selected from the group consistingof: 1, 2, 5, 6, 9, 10, 11, 12, 13 and 14, wherein the peptide promotesmyoblast differentiation; and f) a peptide consisting of an amino acidsequence that differs from SEQ ID NO:19 by at least one substitution,deletion or insertion of an amino acid residue at a position of SEQ IDNO:19 selected from the group consisting of: 1, 2, 5, 6, 9, 10, 11, 12,13 and 16, wherein the peptide promotes myoblast differentiation. 47.The isolated peptide of claim 46, wherein the peptide consists of anamino acid sequence that is at least about 80% identical to an aminoacid sequence selected from the group consisting of: SEQ ID NO:2, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.
 48. The isolatedpeptide of claim 46, wherein the peptide consists of an amino acidsequence that is at least about 90% identical to an amino acid sequenceselected from the group consisting of: SEQ ID NO:2, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18 and SEQ ID NO:19.
 49. The isolated peptide of claim46, wherein the peptide consists of an amino acid sequence selected fromthe group consisting of: SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 andSEQ ID NO:19.
 50. The isolated peptide of claim 46, wherein the peptidecomprises a modification selected from the group consisting ofmethylation, farnesylation, carboxymethylation, geranyl geranylation,glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation, and complexing with a lipidcarrier, or any combination thereof.
 51. A composition comprising theisolated peptide of claim 46 and a pharmaceutically acceptable carrier.52. An isolated nucleic acid molecule comprising a nucleic acid sequenceencoding the peptide of claim
 46. 53. A recombinant nucleic acidmolecule comprising a nucleic acid molecule as set forth in claim 52,operatively linked to a recombinant vector.
 54. A method to promotemyoblast differentiation, comprising administering to a myoblast stemcell the isolated peptide of claim
 46. 55. The method of claim 54,further comprising administering the myoblast stem cell to a patient totreat a cardiac or muscle cell disorder.
 56. The method of claim 54,further comprising administering the myoblast stem cell to an individualto stimulate cardiac or skeletal muscle growth.
 57. A method toregenerate damaged, degenerated or atrophied cardiac and skeletalmyocytes, comprising administering to a patient that has damaged,degenerated or atrophied cardiac or skeletal myocytes, the isolatedpeptide of claim 46, or a composition comprising the peptide.
 58. Themethod of claim 58, comprising contacting a population of myoblast stemcells, myoblasts, or muscle cells with the isolated peptide, andadministering the cells to the patient.
 59. A method to stimulatecardiac or skeletal muscle growth in a mammal, comprising administeringto a mammal the isolated peptide of claim 46, or a compositioncomprising the peptide.
 60. The method of claim 59, comprisingcontacting a population of myoblast stem cells, myoblasts, or musclecells with the isolated peptide, and administering the cells to thepatient.
 61. A method to treat cardiac and skeletal muscle disorders,comprising administering to a patient that has a cardiac or skeletalmuscle disorder the isolated peptide of claim 46, or a compositioncomprising the peptide.
 62. The method of claim 61, wherein saiddisorder is selected from the group consisting of: dilatedcardiomyopathy, Emery-Dreifuss muscular dystrophy, limb-girdle musculardystrophy, partial lipodystrophy, axonal neuropathy, and mandibuloacraldysplasia.
 63. The method of claim 61, comprising contacting apopulation of myoblast stem cells, myoblasts, or muscle cells with theisolated peptide, and administering the cells to the patient.